Recombinant antibodies against hepatitis c virus and methods of obtaining and using same

ABSTRACT

Recombinant antibodies, including chimeric antibodies, specific for hepatitis C (HCV) antigenic proteins are provided. The recombinant antibodies specifically bind to diagnostically relevant regions of HCV proteins and are thus suitable for use, for example, as diagnostic reagents for the detection of HCV, and/or as standardization reagents or positive control reagents in assays for the detection of HCV. The recombinant antibodies can also be used in the treatment or prevention of a HCV infection.

RELATED APPLICATION INFORMATION

This application is a divisional application of U.S. patent applicationSer. No. 11/633,810, filed on Dec. 5, 2006, now allowed, the contents ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates among other things to the field ofrecombinant antibodies and, in particular, to recombinant antibodiesincluding chimeric antibodies against the hepatitis C virus (HCV).

BACKGROUND OF THE INVENTION

Hepatitis C Virus (HCV) is now recognized as being the primary cause oftransfusion associated non A, non B (NANB) hepatitis. HCV is a singlestranded, positive sense RNA virus with similarities to flaviviruses andpestiviruses (Miller R H and Purcell R H. Proc Natl Acad Sci. (1991) 87,2057; Weiner A J, et al. Virology (1990) 180, 842) and is in globaldistribution. HCV contains a plus-strand RNA genome of approximately10,000 nucleotides that encodes a polyprotein precursor of about 3000amino acids. The polyprotein is co- and post-translationally processedby cellular and viral proteases into mature structural andnon-structural proteins. The structural proteins include the coreprotein and the envelope glyco-proteins, E1 and E2. The non-structuralproteins include the NS2-3 auto-protease, the NS3 serine protease, aNTPase/RNA helicase domain in the carboxy terminal two-thirds of NS3,the NS4A polypeptide, the NS4B and NS5A proteins, and the NS5BRNA-dependent RNA polymerase. The HCV genome is heterogeneous and hasbeen classified into six major genotypes (1-6), whose nucleotide anddeduced amino acid sequences vary by about 30% over the entire genome(see, Neville, J. A. et al., J. Clin. Microbiol., 35:3062-3070 (1997)).

Infection with HCV is currently diagnosed by direct detection of viralRNA by PCR or by detection of anti-HCV antibodies (generally to the HCVstructural core protein or non-structural NS3 protein). More recentlyHCV antigen assays have been developed which demonstrate that HCV coreprotein antigens can be detected in a sample sooner than antibodies canbe detected. Studies have shown that the average time from the firstviremic bleed to the first HCV antigen positive bleed is estimated at2.0 days and that the average time to the first HCV antibody positivebleed at 50.8 days (Couroucé A M, et al. Transfusion, (2000) 40,1198-1202).

Currently available HCV test kits that employ anti-HCV antibodies usemonoclonal antibodies. Monoclonal antibodies (mAbs) have been emergingover recent years as increasingly important commercial reagents (see,Smith, K. A., et al., J. Clin. Pathol., 57:912-917 (2004)), especiallyin the area of diagnostics and therapeutics where the exceptionally highdegree of directional binding exhibited by mAbs has contributed to theirsuccess.

Recent developments in recombinant DNA technology have made it possibleto clone the sequences encoding mAbs and to express the antibodies, orfragments of the antibodies, as recombinant proteins. Recombinantantibodies can often be produced more consistently and reliably fromrecombinant constructs than from the original hybridoma. Recombinant DNAtechnology has also allowed the creation of combinations of the heavyand light chain variable regions of a desired non-human mAb with humanconstant regions creating a chimeric antibody (see, for example, U.S.Pat. Nos. 4,816,567 and 6,331,415). The chimeric antibody retains thespecificity and affinity of the original non-human monoclonal antibodybut causes lower human anti-murine antibody (HAMA) responses whenadministered therapeutically and also is capable of reacting in existingdiagnostic assay formats that measure human immunoglobulin.

Monoclonal and recombinant antibodies to HCV have been described. Forexample, U.S. Pat. Nos. 5,595,868 and 7,049,060, and U.S. PatentApplication 2003/0148333 describe monoclonal antibodies to HCV coreprotein; U.S. Patent Application 2004/0208887 describes monoclonalantibodies to HCV E1 protein; U.S. Pat. Nos. 5,308,750 and 7,091,324describe monoclonal antibodies to HCV E2 protein; and U.S. Pat. No.5,753,430 describes monoclonal antibodies to HCV core protein, NS3protein and NS4 protein. Human recombinant antibodies, and specificallyFab fragments derived from human antibodies, specific for HCV NS3protein are described in U.S. Patent Application 2004/0214994. Chimericantibodies to the hypervariable region 1 (HVR1) of HCV have beendescribed (Li, C. and Allain, J-P., J. Gen. Virol., 86:1709-1716(2005)). HVR1 is a highly mutated region of 27 residues at theN-terminus of the E2 protein.

The use of “heterologous” chimeric antibodies as quality controlreagents or calibrators of immunoassays has been described (Hamilton, R.G., Ann. Biol. Clin., 48:473-477 (1990); Hamilton, R. G., Ann. Biol.Clin., 49:242-248 (1991); Naess, L. M., et al., J. of Immunol. Methods,196:41-49; Schuurman, J., et al., J. Allergy Clin. Immunol., 99:545-550(1997)). Heterologous in this context indicates that the chimericantibody binds to an antigen unrelated to the antigen used in the assayfor antibody detection. The use of recombinant mouse-human chimericantibodies, and specifically recombinant mouse-human chimeric antibodiesagainst Toxoplasma gondii, that bind to same or “homologous” antigen ascalibrators or positive controls in assays and kits that measure humanantibodies has also been described (U.S. Pat. No. 6,015,662 and Hackett,J. Jr., et al., J. Clin. Microbiol., 36:1277-1284)).

Moreover, many HCV immunoassays use HCV infected patient blood samplesto prepare a HCV sensitivity panel. Quality control reagents such assensitivity panels are human plasma/serum screened for the presence ofantibodies against specific epitopes. However, the use of humanserum/plasma has several significant disadvantages, including increasedregulatory concerns, difficulty in sourcing large volume with high-titerand specificity, lot-to-lot variability, limitations with respect tocharacterization, and cost.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the invention is to provide recombinant antibodies,including chimeric antibodies, against hepatitis C virus (HCV) and usesthereof. In accordance with one aspect of the present invention, thereis provided an immunodiagnostic reagent comprising one or morerecombinant antibodies, including chimeric antibodies, that are capableof specifically binding a diagnostically relevant region of a HCVprotein. Optionally the one or more recombinant antibodies, includingchimeric antibodies, are selected from the group consisting of arecombinant antibody specific for HCV core protein, a recombinantantibody specific for HCV E2 protein, a recombinant antibody specificfor HCV NS3 protein, a recombinant antibody specific for HCV NS4protein, and a recombinant antibody specific for HCV NS5 protein.

In accordance with another aspect of the invention, there is provided arecombinant antibody, including a chimeric antibody, which specificallybinds to a diagnostically relevant region of a HCV protein wherein thediagnostically relevant region optionally is selected from the groupconsisting of a HCV core protein, a HCV NS3 protein, a HCV NS4 protein,and a HCV NS5 protein.

In accordance with another aspect of the invention, there is provided acell line capable of expressing a chimeric antibody that specificallybinds to a diagnostically relevant region of a HCV protein, wherein thecell line optionally is selected from the group consisting of HCV coreCHO 201-603-486-333, HCV core CHO 14-153-229sc152, HCV NS3 CHO17-903-132sc171, HCV NS4 CHO E99H6C34sc203, and HCV NS5 CHO48-311-271-455. There also is provided a cell line which expresses amouse monoclonal antibody that specifically binds to a diagnosticallyrelevant region of a hepatitis C virus protein, wherein the cell lineoptionally is selected from the group consisting of anti-HCV Core201-603-195, anti-HCV Core 14-153-462, anti-HCV NS3 17-903-127, anti-HCVNS4 E99H6C34, and anti-HCV NS5 48-311-387.

In accordance with another aspect of the present invention, there isprovided a method of standardizing a HCV antibody detection assaycomprising employing as a sensitivity panel an immunodiagnostic reagentoptionally comprising one or more recombinant antibodies, includingrecombinant chimeric antibodies, that are capable of specificallybinding a diagnostically relevant region of a HCV protein. In such apanel, optionally the one or more recombinant antibodies are selectedfrom the group consisting of a recombinant antibody (e.g., a chimericantibody) specific for HCV core protein, a recombinant antibody (e.g., achimeric antibody) specific for HCV E2 protein, a recombinant antibody(e.g., a chimeric antibody) specific for HCV NS3 protein, a recombinantantibody (e.g., a chimeric antibody) specific for HCV NS4 protein, and arecombinant antibody (e.g., a chimeric antibody) specific for HCV NS5protein.

In accordance with another aspect of the present invention, there isprovided a method for detecting the presence of HCV antigens comprisingcontacting a test sample, such as a sample suspected of containing HCVantigens, with an immunodiagnostic reagent comprising one or morerecombinant antibodies, including recombinant chimeric antibodies, whicheach are capable of specifically binding an HCV antigen. Optionally thecontacting is done under conditions that allow formation of recombinantantibody:antigen complexes. Further optionally the method comprisesdetecting any recombinant antibody:antigen complexes formed.

In accordance with another aspect of the present invention, there isprovided a method for detecting the presence of HCV antibodiescomprising contacting a test sample, such as a sample suspected ofcontaining antibodies to HCV, with one or more antigens specific for theHCV antibodies. Optionally this contacting is done under conditions thatallow formation of antigen:antibody complexes, and further optionallythe method comprises detecting the antigen:antibody complexes. Stillfurther, the method optionally comprises employing an immunodiagnosticreagent comprising one or more recombinant antibodies, includingrecombinant chimeric antibodies, wherein each of the antibodies arecapable of specifically binding one of the antigens employed in themethod, e.g., either as a positive control or standardization reagent.

In accordance with another aspect of the present invention, there isprovided a diagnostic kit for the detection of HCV comprising animmunodiagnostic reagent comprising one or more recombinant antibodies,including recombinant chimeric antibodies, that each are capable ofspecifically binding a diagnostically relevant region of a HCV protein.In such a kit, the one or more recombinant antibodies optionally areselected from the group consisting of a recombinant antibody (e.g., achimeric antibody) specific for HCV core protein, a recombinant antibody(e.g., a chimeric antibody) specific for HCV E2 protein, a recombinantantibody (e.g., a chimeric antibody) specific for HCV NS3 protein, arecombinant antibody (e.g., a chimeric antibody) specific for HCV NS4protein, and a recombinant antibody (e.g., a chimeric antibody) specificfor HCV NS5 protein.

In accordance with yet another aspect of the present invention, there isprovided a method of identifying amino acid residues within an HCVprotein epitope that are bound by an antibody that specifically binds tosaid HCV protein epitope. The method optionally comprises the steps of:(a) obtaining a yeast display library comprising a series of peptidesdisplayed on the surface of host cells (i.e., yeast cells), wherein thepeptides have amino acid sequences corresponding to the amino acidsequence of the epitope in which each individual amino acid of theepitope has been sequentially substituted by alanine; (b) contacting theyeast display library with said antibody that is capable specificallybinding the epitope under conditions that permit binding of the antibodyto the epitope, and (c) identifying those peptides displayed on yeastcells which are not bound by said antibody in step (b), wherein anabsence of antibody binding indicates that the yeast displayed peptidecontains an alanine residue at an amino acid position bound by saidantibody in the epitope.

These and other features, aspects, objects, and embodiments of theinvention will become more apparent in the following detaileddescription in which reference is made to the appended drawings that areexemplary of such features, aspects, objects and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a plasmid map of an expression plasmid used in thepreparation of an anti-HCV core chimeric antibody (HCV core201-603-486-333) in one embodiment of the present invention.

FIGS. 2A-D depict the nucleic acid and amino acid sequences in oneembodiment of the present invention of the light and heavy variableregions of an anti-HCV core chimeric antibody (HCV core 201-603-486-333)with underlining denoting the respective complementarity determiningregions (CDRs): FIG. 2A: V_(H) nucleic acid sequence (SEQ ID NO:7); FIG.2B: V_(L) nucleic acid sequence (SEQ ID NO:8); FIG. 2C: V_(H) amino acidsequence (SEQ ID NO:1); and FIG. 2D: V_(L) amino acid sequence (SEQ IDNO:2).

FIG. 3 presents a plasmid map of an expression plasmid used in thepreparation of an anti-HCV NS3 chimeric antibody (HCV NS3 CHO17-903-132sc171) in one embodiment of the present invention.

FIGS. 4A-D depicts the nucleic acid and amino acid sequences in oneembodiment of the present invention of the light and heavy variableregions of an anti-HCV NS3 chimeric antibody (HCV NS3 CHO17-903-132sc171) with underlining denoting the respectivecomplementarity determining regions (CDRs): FIG. 4A: V_(H) nucleic acidsequence (SEQ ID NO:15); FIG. 4B: V_(L) nucleic acid sequence (SEQ IDNO:16); FIG. 4C: V_(H) amino acid sequence (SEQ ID NO:9); and FIG. 4D:V_(L) amino acid sequence (SEQ ID NO:10).

FIG. 5 presents a plasmid map of an expression plasmid used in thepreparation of an anti-HCV NS4 chimeric antibody (HCV NS4 CHOE99H6C34sc203) in one embodiment of the present invention.

FIGS. 6A-D depicts the nucleic acid and amino acid sequences in oneembodiment of the present invention of the light and heavy variableregions of an anti-HCV NS4 chimeric antibody (HCV NS4 CHO E99H6C34sc203)with underlining denoting the respective complementarity determiningregions (CDRs): FIG. 6A: V_(H) nucleic acid sequence (SEQ ID NO:23);FIG. 6B: V_(L) nucleic acid sequence (SEQ ID NO:24); FIG. 6C: V_(H)amino acid sequence (SEQ ID NO:17); and FIG. 6D: V_(L) amino acidsequence (SEQ ID NO:18).

FIG. 7 presents a plasmid map of an expression plasmid used in thepreparation of an anti-HCV NS5 chimeric antibody (HCV NS5 CHO48-311-271-455) in one embodiment of the present invention.

FIGS. 8A-D depicts the nucleic acid and amino acid sequences in oneembodiment of the present invention of the light and heavy variableregions of an anti-HCV NS5 chimeric antibody (HCV NS5 CHO48-311-271-455) with underlining denoting the respective complementaritydetermining regions (CDRs); FIG. 8A: V_(H) nucleic acid sequence (SEQ IDNO:31); FIG. 8B: V_(L) nucleic acid sequence (SEQ ID NO:32); FIG. 8C:V_(H) amino acid sequence (SEQ ID NO:25); and FIG. 8D: V_(L) amino acidsequence (SEQ ID NO:26).

FIGS. 9A-D depicts the epitopes bound by core, NS3, NS4 and NS5 chimericantibodies in one embodiment of the present invention: FIG. 9A: showsthe epitope (SEQ ID NO:34) bound by the anti-HCV core chimeric antibody(HCV core 201-603-486-333); FIG. 9B: shows the epitope (SEQ ID NO:35)bound by the anti-HCV NS3 chimeric antibody (HCV NS3 CHO17-903-132sc171); FIG. 9C: shows the epitope (SEQ ID NO:36) bound by theanti-HCV NS4 chimeric antibody (HCV NS4 CHO E99H6C34sc203); and FIG. 9D:shows the epitope (SEQ ID NO:37) bound by the anti-HCV NS5 chimericantibody (HCV NS5 CHO 48-311-271-455); specific residues bound by therespective chimeric antibodies are underlined and numbered.

FIG. 10 depicts the average chimeric antibody production on a typicalresearch and development scale (i.e. viable cells were between about20-10%) as assessed by HPLC analysis of 3 week old cultures for anti-HCVcore chimeric antibody (“Core”; HCV core 201-603-486-333); anti-HCV NS3chimeric antibody (“NS3”; HCV NS3 CHO 17-903-132sc171); anti-HCV NS4chimeric antibody (“NS4”; HCV NS4 CHO E99H6C34sc203), and anti-HCV NS5chimeric antibody (“NS5”; HCV NS5 CHO 48-311-271-455).

FIGS. 11A-D depicts the nucleic acid and amino acid sequences in oneembodiment of the present invention of the light and heavy variableregions of an anti-HCV core chimeric antibody (HCV core 14-153-229sc152)with underlining denoting the respective complementarity determiningregions (CDRs): FIG. 11A: V_(H) nucleic acid sequence (SEQ ID NO:40);FIG. 11B: V_(L) nucleic acid sequence (SEQ ID NO:41); FIG. 11C: V_(H)amino acid sequence (SEQ ID NO:38); and FIG. 11D: V_(L) amino acidsequence (SEQ ID NO:39).

FIG. 12 presents a plasmid map of an expression plasmid used in thepreparation of an anti-HCV core chimeric antibody (HCV Core14-153-229sc152) in one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention among other things provides recombinantantibodies, including chimeric antibodies, specific for hepatitis C(HCV) antigenic proteins. In accordance with one embodiment of thepresent invention, the recombinant antibodies including chimericantibodies, specifically bind to diagnostically relevant regions of HCVproteins and are thus suitable for use, for example, as diagnosticreagents for the detection of HCV, and/or as standardization reagents orpositive control reagents in assays for the detection of HCV.

The present invention also thus provides for an immunodiagnostic reagentcomprising one or more recombinant antibodies, including chimericantibodies, wherein each antibody is capable of specifically binding adiagnostically relevant region of a HCV protein. The recombinantantibodies can be, for example, chimeric antibodies, humanizedantibodies, antibody fragments, and the like. In another embodiment, theimmunodiagnostic reagent comprises two or more recombinant antibodies,including chimeric antibodies. Optionally the antibodies employed in theimmunodiagnostic reagent are each specific for a different HCV antigenicprotein, such that the immunodiagnostic reagent is capable of detectinga plurality of HCV antigens. Optionally the immunodiagnostic reagentcomprises one or more, or two or more, recombinant antibodies specificfor HCV proteins selected from the group consisting of a recombinantantibody specific for HCV core protein, a recombinant antibody specificfor HCV E2 protein, a recombinant antibody specific for HCV NS3 protein,a recombinant antibody specific for HCV NS4 protein, and a recombinantantibody specific for HCV NS5 protein.

In one embodiment, the present invention provides for the use of theimmunodiagnostic reagent as a standardization reagent in an HCVdetection assay, for instance, in place of human sera. In this context,the immunodiagnostic reagent optionally can be employed, for example, toevaluate and standardize the performance of current and future HCVantigen-detection assays.

In an alternative embodiment, the present invention further provides forthe use of the recombinant antibodies in the treatment or prevention ofa HCV infection.

These and additional embodiments, features, aspects, illustrations, andexamples of the invention are further described in the sections whichfollow. Unless defined otherwise herein, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

Definitions

The term “recombinant antibody” or “recombinant antibodies,” as usedherein, refers to an antibody prepared by one or more steps includingcloning nucleic acid sequences encoding all or a part of one or moremonoclonal antibodies into an appropriate expression vector byrecombinant techniques and subsequently expressing the antibody in anappropriate host cell. The term thus includes, but is not limited to,recombinantly-produced antibodies that are monoclonal antibodies,antibody fragments including fragments of monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multispecific or multivalent structures formed from antibodyfragments, and bifunctional antibodies.

The term “antibody fragment” or “antibody fragments,” as used herein,refers to a portion of an intact antibody comprising the antigen bindingsite or variable region of the intact antibody, wherein the portion isfree of the constant heavy chain domains (i.e., C_(H)2, C_(H)3, andC_(H)4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include, but are not limitedto, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)₂ fragments,Fv fragments, diabodies, single-chain Fv (scFv) molecules, single chainpolypeptides containing only one light chain variable domain, singlechain polypeptides containing the three CDRs of the light chain variabledomain, single chain polypeptides containing only one heavy chainvariable region, and single chain polypeptides containing the three CDRsof the heavy chain variable region.

The term “chimeric antibody” (or “cAb”) as used herein, refers to apolypeptide comprising all or a part of the heavy and light chainvariable regions of an antibody from one host species linked to at leastpart of the antibody constant regions from another host species.

The term “humanized antibody,” as used herein, refers to a polypeptidecomprising a modified variable region of a human antibody wherein aportion of the variable region has been substituted by the correspondingsequence from a non-human species and wherein the modified variableregion is linked to at least part of the constant region of a humanantibody. In one embodiment, the portion of the variable region is allor a part of the complementarity determining regions (CDRs). The termalso includes hybrid antibodies produced by splicing a variable regionor one or more CDRs of a non-human antibody with a heterologousprotein(s), regardless of species of origin, type of protein,immunoglobulin class or subclass designation, so long as the hybridantibodies exhibit the desired biological activity (i.e. the ability tospecifically bind a HCV antigenic protein).

The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e. the bifunctional antibodies have a dual specificity.

The term “diagnostically relevant” as used herein with reference to aregion of a HCV protein refers to a region of the protein the detectionof which, either alone or in combination with other diagnosticallyrelevant regions of HCV, allows detection of at least three of the sixmajor HCV genotypes (see, for example, Neville et al., ibid.). Examplesof diagnostically relevant regions include immunodominant regions knownin the art and regions such as those described herein.

As used herein, the term “epitope”, “epitopes” or “epitopes of interest”refer to a site(s) on any molecule that is recognized and is capable ofbinding to a complementary site(s) on its specific binding partner. Themolecule and specific binding partner are part of a specific bindingpair. For example, an epitope can be a polypeptide, protein, hapten,carbohydrate antigen (such as, but not limited to, glycolipids,glycoproteins or lipopolysaccharides) or polysaccharide and its specificbinding partner, can be, but is not limited to, an antibody. Typicallyan epitope is contained within a larger antigenic fragment (i.e., regionor fragment capable of binding an antibody) and refers to the preciseresidues known to contact the specific binding partner. It is possiblefor an antigenic fragment to contain more than one epitope.

As used herein, “specific” or “specificity” in the context of aninteraction between members of a specific binding pair (e.g., an antigenand antibody) refers to the selective reactivity of the interaction. Thephrase “specifically binds to” and analogous terms thereof refer to theability of antibodies to specifically bind to a HCV protein and notspecifically bind to other entities. Antibodies or antibody fragmentsthat specifically bind to a HCV protein can be identified, for example,by diagnostic immunoassays (e.g., radioimmunoassays (“RIA”) andenzyme-linked immunosorbent assays (“ELISAs”) (See, for example, Paul,ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages332-336 (1989)), BIAcore® (Sweden), KinExA® (Kinetic Exclusion Assay,available from Sapidyne Instruments (Boise, Id.)) or other techniquesknown to those of skill in the art.

As used herein, the term “equilibrium dissociation constant” or “K_(D)”as used interchangeably, refer to the value obtained by dividing thedisassociation rate constant (k_(off)) by the association rate constant(k_(on)). The association rate constant, the disassociation rateconstant and the equilibrium dissociation constant are used to representthe binding affinity of an antibody to an antigen. Methods fordetermining these constants are well known in the art. For example, aBiacore® or KinExA® assay can be used.

As described herein, immunoassays incorporate “quality control reagents”that include but are not limited to, e.g., calibrators, controls, andsensitivity panels. A “calibrator” or “standard” typically is used(e.g., one or more, or a plurality) in order to establish calibration(standard) curves for interpolation of antibody concentration.Optionally, a single calibrator may be used near the positive/negativecutoff Multiple calibrators (i.e., more than one calibrator or a varyingamount of calibrator(s)) can be used in conjunction so as to comprise a“sensitivity panel. A “positive control” is used to establish assayperformance characteristics and is a useful indicator of the integrityof the reagents (e.g., antigens).

The term “substantially identical,” as used herein in relation to anucleic acid or amino acid sequence indicates that, when optimallyaligned, for example using the methods described below, the nucleic acidor amino acid sequence shares at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98% or atleast about 99% sequence identity with a defined second nucleic acid oramino acid sequence (or “reference sequence”). “Substantial identity”may be used to refer to various types and lengths of sequence, such asfull-length sequence, epitopes or immunogenic peptides, functionaldomains, coding and/or regulatory sequences, exons, introns, promoters,and genomic sequences. Percent identity between two amino acid ornucleic acid sequences can be determined in various ways that are withinthe skill of a worker in the art, for example, using publicly availablecomputer software such as Smith Waterman Alignment (Smith, T. F. and M.S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman,Advances in Applied Mathematics, 482-489 (1981)) as incorporated intoGeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequenceand Structure, Dayhof, M. O., Ed pp 353-358; BLAST program (Basic LocalAlignment Search Tool (Altschul, S. F., W. Gish, et al. (1990) J MolBiol 215: 403-10), and variations thereof including BLAST-2, BLAST-P,BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, and Megalign(DNASTAR) software. In addition, those skilled in the art can determineappropriate parameters for measuring alignment, including algorithmsneeded to achieve maximal alignment over the length of the sequencesbeing compared. In general, for amino acid sequences, the length ofcomparison sequences will be at least about 10 amino acids. One skilledin the art will understand that the actual length will depend on theoverall length of the sequences being compared and may be at least about20, at least about 30, at least about 40, at least about 50, at leastabout 60, at least about 70, at least about 80, at least about 90, atleast about 100, at least about 110, at least about 120, at least about130, at least about 140, at least about 150, at least about 200, atleast about 250, at least about 300, or at least about 350 amino acids,or it may be the full-length of the amino acid sequence. For nucleicacids, the length of comparison sequences will generally be at leastabout 25 nucleotides, but may be at least about 50, at least about 100,at least about 125, at least about 150, at least about 200, at leastabout 250, at least about 300, at least about 350, at least about 400,at least about 450, at least about 500, at least about 550, at leastabout 600, at least about 650, at least about 700, at least about 800,at least about 900, or at least about 1000 nucleotides, or it may be thefull-length of the nucleic acid sequence.

The terms “corresponding to” or “corresponds to” indicate that a nucleicacid sequence is identical to all or a portion of a reference nucleicacid sequence. The term “complementary to” is used herein to indicatethat the nucleic acid sequence is identical to all or a portion of thecomplementary strand of a reference nucleic acid sequence. Forillustration, the nucleic acid sequence “TATAC” corresponds to areference sequence “TATAC” and is complementary to a reference sequence“GTATA.”

Unless otherwise specified herein, all nucleic acid sequences arewritten in a 5′ to 3′ direction, and all amino acid sequences arewritten in an amino- to carboxy-terminus direction.

As used herein, the term “about” refers to approximately a +/−10%variation from the stated value. It is to be understood that such avariation is always included in any given value provided herein, whetheror not it is specifically referred to.

1. Immunodiagnostic Reagent

The immunodiagnostic reagent of the present invention comprises one ormore recombinant antibodies, including recombinant chimeric antibodies,that specifically bind to a diagnostically relevant region of a HCVprotein. In one embodiment, therefore, the immunodiagnostic reagentprovided by the present invention comprises a single chimeric antibodycapable of specifically binding a diagnostically relevant region of aHCV protein. In other embodiments, the immunodiagnostic reagentcomprises a plurality of chimeric antibodies, each capable ofspecifically binding a diagnostically relevant region of a HCV protein(e.g., either the same region, or a different region). One or more ofthe plurality of chimeric antibodies may be capable of specificallybinding a diagnostically relevant region of the same HCV protein.Alternatively, each of the plurality of chimeric antibodies mayspecifically bind a diagnostically relevant region of a different HCVprotein.

In one embodiment of the present invention, the immunodiagnostic reagentis capable of detecting a plurality of HCV antigens and optionallycomprises two or more recombinant antibodies, each capable ofspecifically binding a different HCV antigenic protein. In a furtherembodiment, the immunodiagnostic reagent optionally comprises three ormore recombinant antibodies, each capable of specifically binding adifferent HCV antigenic protein. In another embodiment, theimmunodiagnostic reagent optionally comprises four or more recombinantantibodies, each capable of specifically binding a different HCVantigenic protein.

The recombinant antibodies comprised by the immunodiagnostic reagent canoptionally be modified, for example, for detection purposes, forimmobilization onto a solid support, to improve stability and/or toimprove pharmacokinetic properties, or by other means such as is knownin the art.

2. HCV Antigens and Diagnostically Relevant Regions Thereof

HCV contains a plus-strand RNA genome of approximately 10,000nucleotides that encodes a polyprotein precursor of 3011 amino acids.The polyprotein is co- and post-translationally processed by cellularand viral proteases into the mature structural and nonstructuralproteins. The structural proteins include the core protein (amino acids1 to 192 of the polyprotein) and the envelope glyco-proteins, E1 (fromamino acid 193 to 384 of the polyprotein) and E2 (from amino acid 385 to747 of the polyprotein). The nonstructural (NS) proteins 2-5B includethe NS2-3 auto-protease (from amino acid 811 to 1027 of thepolyprotein), the NS3 serine protease (from amino acid 1028 to 1658 ofthe polyprotein), a NTPase/RNA helicase domain in the carboxy terminaltwo-thirds of NS3, the NS4A polypeptide (from amino acid 1659 to 1712 ofthe polyprotein), the NS4B and NS5A proteins (from amino acid 1713 to1973, and amino acid 1974 to 2421 of the polyprotein, respectively), andthe NS5B RNA-dependent RNA polymerase (from amino acid 2422 to 3011 ofthe polyprotein).

Diagnostically relevant regions of many of these proteins have beendetermined. For core protein, the region defined, for example, by aminoacids 1-150, and sub-regions thereof such as amino acids 2 to 120, aminoacids 10 to 53, and amino acids 1 to 50, have been determined to bediagnostically relevant. For the NS3 protein, the region defined by, forexample, amino acids 1192 to 1457 has been determined to bediagnostically relevant. For the NS4 protein, the regions defined by,for example, amino acids 1920 to 1935 and amino acids 1676 to 1931, andsub-regions thereof such as amino acids 1696-1931 and amino acids 1694to 1735, have been determined to be diagnostically relevant. For NS5,the region defined by, for example, amino acids 2054 to 2995, andsub-regions thereof such as amino acids 2188 to 2481 and amino acids2212 to 2313 (see Dou, X-G., et al., J. Clin. Microbiol. (2002)40:61-67), have been determined to be diagnostically relevant. For E1and E2 the regions defined by amino acids 303-320, and amino acids405-444, respectively, have been determined to be diagnosticallyrelevant. Other examples include, but are not limited to, the regiondefined by amino acids 1192 to 1931 (spanning NS3 and NS4), the regiondefined by amino acids 1569 to 1931 (spanning NS3 and NS4) and theregion defined by amino acids 1932 to 2191 (spanning NS4 and NS5).

3. Recombinant Antibodies

The recombinant antibodies of the present invention compriseantigen-binding regions derived from the V_(H) and/or V_(L) domains of anative antibody capable of specifically binding to a HCV antigenicprotein. The recombinant antibody can be, for example, arecombinantly-produced monoclonal antibody, a fragment of a monoclonalantibody, a chimeric antibody, a humanized antibody, a multispecific ormultivalent structure formed from an antibody fragment, or abifunctional antibody.

In one embodiment, optionally the recombinant antibody is a chimericantibody that retains the mouse monoclonal antibody specificity andaffinity and reacts in an immunoassay format that measures humanimmunoglobulin. Optionally the mouse-human chimeric antibody is directedagainst the HCV Core, NS3, NS4, and/or NS5 antigen. Optionally such achimeric antibody reacts in an existing immunoassay format including butnot limited to Abbott Laboratories' HCV assay for EIA (Bead), AxSYM®,ARCHITECT® and PRISM® platforms.

The antigen-binding region comprised by the recombinant antibody caninclude the entire V_(H) and/or V_(L) sequence from the native antibody,or it can comprise one or more portions thereof, such as the CDRs,together with sequences derived from one or more other antibodies. Inone embodiment, the recombinant antibody comprises the full-length V_(H)and V_(L) sequences of the native antibody.

The native antibody from which the antigen-binding regions are derivedis generally a vertebrate antibody. For example, the native antibody canbe a rodent (e.g. mouse, hamster, rat) antibody, a chicken antibody, arabbit antibody, a canine antibody, a feline antibody, a bovineantibody, an equine antibody, a porcine antibody, an ape (e.g.chimpanzee) antibody, or a human antibody. The source of the antibody isbased primarily on convenience. In one embodiment, the native antibodyis a non-human antibody.

The recombinant antibody also can include one or more constant regions,for example, the C_(L), C_(H)1, hinge, C_(H)2, C_(H)3, and/or C_(H)4regions, derived from the same native antibody or from a differentantibody. The constant region(s) can be derived from an antibody fromone of a number of vertebrate species, including but not limited to,those listed above. In one embodiment of the present invention, therecombinant antibody comprises at least one constant region. In anotherembodiment, the recombinant antibody comprises one or more constantregions that are derived from a human antibody. In a specific embodimentof the present invention, the recombinant antibody comprises thevariable region of a non-human antibody linked to the constant region ofa human antibody.

The constant region(s) comprised by the recombinant antibody can bederived from one or more immunoglobulin classes or isotypes, for examplefor constant regions derived from human immunoglobulins, the constantregion can be derived from one or more of an IgM, IgD, IgG1, IgG2, IgG3,IgG4, IgA1, IgA2 or IgE constant region. When the constant regioncomprises a region derived from an IgG light chain, this may be derivedfrom a kappa chain or a lambda chain. The recombinant antibody maycomprise sequences from more than one class or isotype. Selection ofparticular constant domains to optimize the desired function of therecombinant antibody is within the ordinary skill in the art. In oneembodiment of the present invention, the recombinant antibody comprisesone or more constant domains derived from an IgG. In another embodiment,the recombinant antibody comprises regions from both the heavy and lightchains of an IgG constant domain.

In one embodiment of the present invention, the antigen-binding regionsare derived from a native antibody that specifically binds to an epitopewithin a diagnostically relevant region of a HCV antigenic protein.Non-limiting examples of diagnostically relevant regions of HCV proteinsare provided above. In another embodiment, the antigen-binding regionsare derived from a native antibody that specifically binds to an epitopewithin the region defined by amino acids 1-150 of core protein, theregion defined by amino acids 1192 to 1457 of the NS3 protein, theregions defined by amino acids 1920 to 1935 or amino acids 1676 to 1931of the NS4 protein, or the region defined by amino acids 2054 to 2995 ofthe NS5 protein. In a further embodiment, the antigen-binding regionsare derived from a native antibody that specifically binds to an epitopewithin the region of the core protein defined by amino acids 1-50 of theHCV polyprotein, within the region of the NS3 protein defined by aminoacids 1192-1457 of the HCV polyprotein, within the region of the NS4protein defined by amino acids 1676-1931 of the HCV polyprotein, orwithin the region of the NS5 protein defined by amino acids 1932-2191 oramino acids 2188-2481 of the HCV polyprotein. In another embodiment, theantigen-binding regions are derived from a native antibody thatspecifically binds to a region of the core protein as set forth in SEQID NO:33 (GGVYL) or SEQ ID NO:34 (GGQIVGGVYLLPR), a region of the NS3protein as set forth in SEQ ID NO:35 (AKAVDFVPVESLETTMRSPVFTDNSSP), aregion of the NS4 protein as set forth in SEQ ID NO:36(PAIIPDREVLYREFDEMEECSQ), or a region of the NS5 protein as set forth inSEQ ID NO:37 (AESYSSMPPLEGEPGDPDLSDGSWSTV).

In a specific embodiment of the present invention, the antigen-bindingregions of the recombinant antibody comprise an amino acid sequencesubstantially identical to all or a portion of the V_(H) or V_(L)sequence as set forth in any one of SEQ ID NOs: 1, 2, 9, 10, 17, 18, 25,26, 38 and 39 (see Table 1). In another embodiment, the antigen-bindingregions of the recombinant antibody comprise the complementaritydetermining regions (CDRs; i.e. CDR1, CDR2 and CDR3) of a V_(H) or V_(L)sequence. In a specific embodiment, the antigen-binding regions of therecombinant antibody comprise an amino acid sequence substantiallyidentical to one or more of the CDRs (see Table 1): as set forth in SEQID NOs:42, 43 and 44; as set forth in SEQ ID NOs:45, 46 and 47; as setforth in SEQ ID NOs:48, 49 and 50; as set forth in SEQ ID NOs:51, 52 and53; as set forth in SEQ ID NOs:54, 55 and 56; as set forth in SEQ IDNOs:57, 58 and 59; as set forth in SEQ ID NOs:60, 43 and 61; as setforth in SEQ ID NOs:62, 58 and 63; as set forth in SEQ ID NOs:64, 65 and66; or as set forth in SEQ ID NOs:67, 46 and 68.

TABLE 1 Exemplary V_(H) and V_(L) Sequences SEQ HCV ID Protein NOSequence V_(H) or V_(L) Specificity 1 QIQLVQSGPELQKPGKTVKISCKTSGYTFTDYPV_(H) Core MHWVKQAPGKGLKWMGWINTETGEPTRVDDFKGRFAFSLETSASTAYLQINNLKDEDTATYFC ARGGGVRRQVMDYWGQGTSVTVSS 2DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSR V_(L) CoreTRKNYLVWYQQKPGQSPKLLIYWASTRDSGVP DRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIKR 38 QIQLVQSGPELKKPGETVKISCKASGYTFTNYG V_(H) CoreMNWVKQAPGKGLKWMGWINTNTGEPTYAEE FKGRFAFSLETSAITAYLQINNLKNEDTATYFCARAGGDYYDSSYDYAMDYWGQGTSVTVSS 39 DIVLTQSPASLAVSLGQRATISCKASQSVDYDG V_(L)Core DSYMNWYQQKPGQPPKLLIYAASNLESGIPAR FSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKR 9 EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYY V_(H) NS3MYWVRQTPEKRLEWAAYISNGAGSTYYPDTV KGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARGLWDGLDYWGQGTTLTVSS 10 DVVMAQTPLSLPVSLGDQASISCRSSQSLVHSN V_(L) NS3GNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKR 17 QIQLVQSGPELKKPGETVKISCKASGYTFTDYS V_(H) NS4MHWVNQAPGKGLKWMGWINTETGEPTYADD FKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRGGTGYWGQGTTLTVSS 18 DVVMTQTPLSLPVSLGDQASISCRSSQSLVYSN V_(L) NS4GNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKR 25 EVQLQQSGAELVKPGASVKLSCTASGFNIKDT V_(H) NS5YMHWVKQRPEQGLEWIGRIDPANGNTKYDPK FQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARSREFAYWGQGTLVTVSA 26 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSS V_(L) NS5NQKNYLAWYQQKPGQSPKLLIYWASTRESGVP DRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKR 42 GYTFTDYP CDR1 V_(H) Core 43 INTETGEP CDR2 V_(H) Core44 ARGGGVRRQVMDY CDR3 V_(H) Core 45 QSLLNSRTRKNY CDR1 V_(L) Core 46 WASCDR2 V_(L) Core 47 KQSYNLYT CDR3 V_(L) Core 48 GYTFTNYG CDR1 V_(H) Core49 INTNTGEP CDR2 V_(H) Core 50 ARAGGDYYDSSYDYAMDY CDR3 V_(H) Core 51QSVDYDGDSY CDR1 V_(L) Core 52 AAS CDR2 V_(L) Core 53 QQSNEDPWTCDR3 V_(L) Core 54 GFTFSDYY CDR1 V_(H) NS3 55 ISNGAGST CDR2 V_(H) NS3 56ARGLWDGLDY CDR3 V_(H) NS3 57 QSLVHSNGNTY CDR1 V_(L) NS3 58 KVSCDR2 V_(L) NS3 59 SQSTHVPYT CDR3 V_(L) NS3 60 GYTFTDYS CDR1 V_(H) NS4 43INTETGEP CDR2 V_(H) NS4 61 TRGGTGY CDR3 V_(H) NS4 62 QSLVYSNGNTYCDR1 V_(L) NS4 58 KVS CDR2 V_(L) NS4 63 SQSTHVPWT CDR3 V_(L) NS4 64GFNIKDTY CDR1 V_(H) NS5 65 IDPANGNT CDR2 V_(H) NS5 66 ARSREFAYCDR3 V_(H) NS5 67 QSLLYSSNQKNY CDR1 V_(L) NS5 46 WAS CDR2 V_(L) NS5 68QQYYSYPLT CDR3 V_(L) NS5

In one embodiment of the present invention, the antigen-binding regionsof the recombinant antibody comprise an amino acid sequencesubstantially identical to all or a portion of the amino acid sequenceencoded by any one of SEQ ID NOs:7, 8, 15, 16, 23, 24, 31, 32, 40 or 41(see Table 2). In another embodiment, the antigen-binding regions of therecombinant antibody comprise a nucleic acid sequence encoding thecomplementarity determining regions (CDRs; i.e. CDR1, CDR2 and CDR3) ofa V_(H) or V_(L) sequence. In a specific embodiment, the antigen-bindingregions of the recombinant antibody comprise CDRs having an amino acidsequence substantially identical to the amino acid sequences encoded byone or more of SEQ ID NOs:69, 70 and 71; one or more of SEQ ID NOs:72,73 and 74; one or more of SEQ ID NOs:75, 76 and 77; one or more of SEQID NOs:78, 79 and 80; one or more of SEQ ID NOs:81, 82 and 83; one ormore of SEQ ID NOs:84, 85 and 86; one or more of SEQ ID NOs:87, 70 and88; one or more of SEQ ID NOs:89, 85 and 90; one or more of SEQ IDNOs:91, 92 and 93; or one or more of SEQ ID NOs:94, 73 and 95 (see Table2).

In another specific embodiment of the present invention, theantigen-binding regions of the recombinant antibody comprise an aminoacid sequence encoded by a nucleic acid sequence substantially identicalto all or a portion of the sequence as set forth in any one of SEQ IDNOs:7, 8, 15, 16, 23, 24, 31, 32, 40 or 41. In a further specificembodiment, the antigen-binding regions of the recombinant antibodycomprise CDRs encoded by nucleic acid sequences substantially identicalto the sequences as set forth in SEQ ID NOs:69, 70 and 71; as set forthin SEQ ID NOs:72, 73 and 74; as set forth in SEQ ID NOs:75, 76 and 77;as set forth in SEQ ID NOs:78, 79 and 80; as set forth in SEQ ID NOs:81,82 and 83; as set forth in SEQ ID NOs:84, 85 and 86; as set forth in SEQID NOs:87, 70 and 88; as set forth in SEQ ID NOs:89, 85 and 90; as setforth in SEQ ID NOs:91, 92 and 93; or as set forth in SEQ ID NOs:94, 73and 95.

TABLE 2Exemplary Nucleic Acid Sequences Encoding V_(H) and V_(L) Sequences SEQHCV ID Encoding Protein NO Sequence V_(H) or V_(L) Specificity 7CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGCAG V_(H) CoreAAGCCTGGAAAGACAGTCAAGATCTCCTGCAAGACTTCTGGTTATACCTTCACAGACTATCCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACACGTGTAGATGACTTCAAGGGACGTTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAGATGAGGACACGGCCACATATTTCTGCGCTAGAGGGGGTGGGGTCCGACGCCAGGTTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 8GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTG V_(L) CoreTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAATAGTAGAACCCGAAAGAACTACTTGGTTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGATTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAAC 40CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAG V_(H) CoreAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAAGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCAACACTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCATCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGCGGGGGGAGATTACTACGATAGTAGCTACGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGT CACCGTCTCCTCA 41GACATTGTGCTGACCCAATCCCCAGCTTCTTTGGCTG V_(L) CoreTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAACGT 15GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTG V_(H) NS3CAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTATATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGCCGCATACATTAGTAATGGTGCTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGGCCTCTGGGACGGCCTTGACTACTGGGGCCAA GGCACCACTCTCACAGTCTCCTCG 16GATGTTGTGATGGCCCAAACTCCACTCTCCCTGCCTG V_(L) NS3TCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGA AATAAAACGT 23CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAG V_(H) NS4AAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCACTGGGTGAACCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTACTAGGGGAGGCACGGGCTACTGGGGCCAAGGCACCACTC TCACAGTCTCCTCA 24GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTG V_(L) NS4TCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTATACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAACGG 31GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTG V_(H) NS5AAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGATCGCGGGAGTTTGCTTACTGGGGCCAAGGGAC TCTGGTCACTGTCTCTGCA 32GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTG V_(L) NS5TGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCT GGAGCTGAAACGG 69GGTTATACCTTCACAGACTATCCA CDR1 V_(H) Core 70 ATAAACACTGAGACTGGTGAGCCACDR2 V_(H) Core 71 GCTAGAGGGGGTGGGGTCCGACGCCAGGTTATGGAC CDR3 V_(H) CoreTAC 72 CAGAGTCTGCTCAATAGTAGAACCCGAAAGAACTAC CDR1 V_(L) Core 73 TGGGCATCCCDR2 V_(L) Core 74 AAGCAATCTTATAATCTGTACACG CDR3 V_(L) Core 75GGGTATACCTTCACAAACTATGGA CDR1 V_(H) Core 76 ATAAACACCAACACTGGAGAGCCACDR2 V_(H) Core 77 GCAAGAGCGGGGGGAGATTACTACGATAGTAGCTAC CDR3 V_(H) CoreGACTATGCTATGGACTAC 78 CAAAGTGTTGATTATGATGGTGATAGTTAT CDR1 V_(L) Core 79GCTGCATCC CDR2 V_(L) Core 80 CAGCAAAGTAATGAGGATCCGTGGACG CDR3 V_(L) Core81 GGATTCACTTTCAGTGACTATTAT CDR1 V_(H) NS3 82 ATTAGTAATGGTGCTGGTAGCACCCDR2 V_(H) NS3 83 GCAAGAGGCCTCTGGGACGGCCTTGACTAC CDR3 V_(H) NS3 84CAGAGCCTTGTACACAGTAATGGAAACACCTAT CDR1 V_(L) NS3 85 AAAGTTTCC CDR2 V_(L)NS3 86 TCTCAAAGTACACATGTTCCGTACACG CDR3 V_(L) NS3 87GGTTATACCTTCACAGACTATTCA CDR1 V_(H) NS4 70 ATAAACACTGAGACTGGTGAGCCACDR2 V_(H) NS4 88 ACTAGGGGAGGCACGGGCTAC CDR3 V_(H) NS4 89CAGAGCCTTGTATACAGTAATGGAAACACCTAT CDR1 V_(L) NS4 85 AAAGTTTCC CDR2 V_(L)NS4 90 TCTCAAAGTACACATGTTCCGTGGACG CDR3 V_(L) NS4 91GGCTTCAACATTAAAGACACCTAT CDR1 V_(H) NS5 92 ATTGATCCTGCGAATGGTAATACTCDR2 V_(H) NS5 93 GCTAGATCGCGGGAGTTTGCTTAC CDR3 V_(H) NS5 94CAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC CDR1 V_(L) NS5 73 TGGGCATCCCDR2 V_(L) NS5 95 CAGCAATATTATAGCTATCCGCTCACG CDR3 V_(L) NS5

The amino acid sequence of recombinant antibody need not correspondprecisely to the parental sequences, i.e. it may be a “variantsequence.” For example, depending in the domains comprised by therecombinant antibody, one or more of the V_(L), V_(H), C_(L), C_(H)1,hinge, C_(H)2, C_(H)3, and C_(H)4, as applicable, may be mutagenized bysubstitution, insertion or deletion of one or more amino acid residuesso that the residue at that site does not correspond to either theparental (or reference) sequence. One skilled in the art willappreciate, however, that such mutations will not be extensive and willnot dramatically affect binding of the recombinant antibody to itstarget HCV protein. In accordance with the present invention, when arecombinant antibody comprises a variant sequence, the variant sequenceis at least about 70% identical to the reference sequence. In oneembodiment, the variant sequence is at least about 75% identical to thereference sequence. In other embodiments, the variant sequence is atleast about 80%, at least about 85% or at least about 90% identical tothe reference sequence. In a specific embodiment, the reference sequencecorresponds to a sequence as set forth in any one of SEQ ID NOs: 1, 2,9, 10, 17, 18, 25, 26, 38 and 39. In another embodiment, the referencesequence corresponds to a sequence as set forth in any one of SEQ IDNOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67 and 68.

Generally, when the recombinant antibody comprises a variant sequencethat contains one or more amino acid substitutions, they are“conservative” substitutions. A conservative substitution involves thereplacement of one amino acid residue by another residue having similarside chain properties. As is known in the art, the twenty naturallyoccurring amino acids can be grouped according to the physicochemicalproperties of their side chains. Suitable groupings include alanine,valine, leucine, isoleucine, proline, methionine, phenylalanine andtryptophan (hydrophobic side chains); glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine (polar, uncharged sidechains); aspartic acid and glutamic acid (acidic side chains) andlysine, arginine and histidine (basic side chains). Another grouping ofamino acids is phenylalanine, tryptophan, and tyrosine (aromatic sidechains). A conservative substitution involves the substitution of anamino acid with another amino acid from the same group.

Thus, the present invention in other embodiments further providesisolated polypeptides corresponding to novel recombinant antibodysequences disclosed herein. Optionally the isolated polypeptidecomprises a portion of a recombinant (e.g., chimeric) antibody thatspecifically binds to a diagnostically relevant region of a HCV proteinselected from the group consisting of HCV core protein, HCV NS3 protein,HCV NS4 protein, and HCV NS5 protein. In one embodiment the polypeptidecomprises a V_(H) region selected from the group consisting of a V_(H)region comprising an amino acid sequence substantially identical to thesequence as set forth in any one or more of SEQ ID NOs:1, 9, 17, 25, and38. In another embodiment the polypeptide comprises a V_(H) regioncomprising complementarity determining region sequences that aresubstantially identical to: one or more of the sequences set forth inSEQ ID NOs: 42, 43 and 44; one or more of the sequences set forth in SEQID NOs:48, 49 and 50; one or more of the sequences set forth in SEQ IDNOs: 54, 55 and 56; one or more of the sequences set forth in SEQ IDNOs: 60, 43 and 61; and/or to one or more of the sequences set forth inSEQ ID NOs:64, 65 and 66. In another embodiment the polypeptidecomprises a V_(L) region comprising an amino acid sequence that issubstantially identical to the sequence as set forth in any one or moreof SEQ ID NOS:2, 10, 18, 26, and 39. In still another embodiment thepolypeptide comprises a V_(L) region comprising complementaritydetermining region sequences that are substantially identical to: one ormore of the sequences set forth in SEQ ID NOs: 45, 46 and 47; one ormore of the sequences set forth in SEQ ID NOs: 51, 52 and 53; one ormore of the sequences set forth in SEQ ID NOs: 57, 58 and 59; one ormore of the sequences set forth in SEQ ID NOs: 62, 58 and 63; and/or oneor more of the sequences set forth in SEQ ID NOs: 67, 46 and 68.

In still another embodiment, the polypeptide comprises a V_(H) regionselected from the group consisting of a V_(H) region comprising an aminoacid sequence substantially identical to the sequence encoded by any oneor more of SEQ ID NOs: 7, 15, 23, 31, and 40. In yet another embodiment,the polypeptide comprises a V_(H) region comprising complementaritydetermining region sequences that are substantially identical to: one ormore of the sequences encoded by SEQ ID NOs: 69, 70 and 71; one or moreof the sequences encoded by SEQ ID NOs:75, 76 and 77; one or more of thesequences encoded by SEQ ID NOs: 81, 82 and 83; one or more of thesequences encoded by SEQ ID NOs: 87, 70 and 88; one or more of thesequences encoded by SEQ ID NOs: 91, 92 and 93. In another embodiment,the polypeptide comprises a V_(L) region selected from the groupconsisting of a V_(L) region comprising an amino acid sequencesubstantially identical to the sequence encoded by any one or more ofSEQ ID NOs:8, 16, 24, 32 and 41. In still yet another embodiment, thepolypeptide comprises a V_(L) region comprising complementaritydetermining region sequences substantially identical to one or more of:the sequences encoded by SEQ ID NOs: 89, 85 and 90; the sequencesencoded by SEQ ID NOs: 84, 85 and 86; the sequences encoded by SEQ IDNOs: 72, 73 and 74; and/or the sequences encoded by SEQ ID NOs: 78, 79and 80; and/or one or more of the sequences encoded by SEQ ID NOs: 94,73 and 95.

Likewise, the nucleic acid sequence encoding the variable region(s) neednot correspond precisely to the parental reference sequence but may varyby virtue of the degeneracy of the genetic code and/or such that itencodes a variant amino acid sequence as described above. In oneembodiment of the present invention, therefore, the nucleic acidsequence encoding a variable region of the recombinant antibody is atleast about 70% identical to the reference sequence. In anotherembodiment, the nucleic acid sequence encoding a variable region of therecombinant antibody is at least about 75% identical to the referencesequence. In other embodiments, the nucleic acid sequence encoding avariable region of the recombinant antibody is at least about 80%, atleast about 85% or at least about 90% identical to the referencesequence. In a specific embodiment, the reference sequence correspondsto a sequence as set forth in any one of SEQ ID NOs:7, 8, 15, 16, 23,24, 31, 32, 40 or 41. In another embodiment, the reference sequencecorresponds to a sequence as set forth in any one of SEQ ID NOs: 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94 and 95.

Thus, the present invention in other embodiments further providesisolated polynucleotides which encode novel recombinant antibodysequences (including chimerical antibody sequences) disclosed herein.Optionally the isolated polynucleotide encodes a portion of arecombinant (e.g., chimeric) antibody that specifically binds to adiagnostically relevant region of a HCV protein selected from the groupconsisting of HCV core protein, HCV NS3 protein, HCV NS4 protein, andHCV NS5 protein. In one embodiment the polynucleotide encodes a V_(H)region selected from the group consisting of a V_(H) region comprisingan amino acid sequence substantially identical to the sequence as setforth in any one or more of SEQ ID NOs:1, 7, 9, 17, 25, and 38. Inanother embodiment the polynucleotide encodes a V_(H) region comprisingcomplementarity determining region sequences that are substantiallyidentical to: one or more of the sequences set forth in SEQ ID NOs: 42,43 and 44; one or more of the sequences set forth in SEQ ID NOs:48, 49and 50; one or more of the sequences set forth in SEQ ID NOs: 54, 55 and56; one or more of the sequences set forth in SEQ ID NOs: 60, 43 and 61;and/or to one or more of the sequences set forth in SEQ ID NOs:64, 65and 66. In another embodiment the polynucleotide encodes a V_(L) regioncomprising an amino acid sequence that is substantially identical to thesequence as set forth in any one or more of SEQ ID NOS:2, 10, 18, 26,and 39. In still another embodiment the polynucleotide encodes a V_(L)region comprising complementarity determining region sequences that aresubstantially identical to: one or more of the sequences set forth inSEQ ID NOs: 45, 46 and 47; one or more of the sequences set forth in SEQID NOs: 51, 52 and 53; one or more of the sequences set forth in SEQ IDNOs: 57, 58 and 59; one or more of the sequences set forth in SEQ IDNOs: 62, 58 and 63; and/or one or more of the sequences set forth in SEQID NOs: 67, 46 and 68.

In still another embodiment, the polynucleotide encodes a V_(H) regionselected from the group consisting of a V_(H) region comprising an aminoacid sequence substantially identical to the sequence encoded by any oneor more of SEQ ID NOs: 7, 15, 23, 31 and 40. In yet another embodiment,the polynucleotide encodes a V_(H) region comprising complementaritydetermining region sequences that are substantially identical to: one ormore of the sequences encoded by SEQ ID NOs: 69, 70 and 71; one or moreof the sequences encoded by SEQ ID NOs:75, 76 and 77; one or more of thesequences encoded by SEQ ID NOs: 81, 82 and 83; one or more of thesequences encoded by SEQ ID NOs: 87, 70 and 88; one or more of thesequences encoded by SEQ ID NOs: 91, 92 and 93. In another embodiment,the polynucleotide encodes a V_(L) region selected from the groupconsisting of a V_(L) region comprising an amino acid sequencesubstantially identical to the sequence encoded by any one or more ofSEQ ID NOs:8, 16, 24, 32 and 41. In still yet another embodiment, thepolynucleotide encodes a V_(L) region comprising complementaritydetermining region sequences substantially identical to one or more of:the sequences encoded by SEQ ID NOs: 89, 85 and 90; the sequencesencoded by SEQ ID NOs: 84, 85 and 86; the sequences encoded by SEQ IDNOs: 72, 73 and 74; and/or the sequences encoded by SEQ ID NOs: 78, 79and 80; and/or one or more of the sequences encoded by SEQ ID NOs: 94,73 and 95.

In one embodiment, the antibodies can be further modified to reduce theimmunogenicity to a human relative to the native antibody by mutatingone or more amino acids in the non-human portion of the antibody thatare potential epitopes for human T-cells in order to eliminate or reducethe immunogenicity of the antibody when exposed to the human immunesystem. Suitable mutations include, for example, substitutions,deletions and insertions of one or more amino acids.

In one embodiment, the recombinant antibodies of the present inventioncan be further modified for immobilization onto a suitable solid phase.Immobilization can be achieved through covalent or non-covalent (forexample, ionic, hydrophobic, or the like) attachment to the solid phase.Suitable modifications are known in the art and include the addition ofa functional group or chemical moiety to either the C-terminus or theN-terminus of one of the amino acid sequences comprised by therecombinant antibody to facilitate cross-linking or attachment of therecombinant antibody to the solid support. Exemplary modificationsinclude the addition of functional groups such asS-acetylmercaptosuccinic anhydride (SAMSA) or S-acetyl thioacetate(SATA), or addition of one or more cysteine residues to the N- orC-terminus of the amino acid sequence. Other cross-linking reagents areknown in the art and many are commercially available (see, for example,catalogues from Pierce Chemical Co. and Sigma-Aldrich). Examplesinclude, but are not limited to, diamines, such as 1,6-diaminohexane;dialdehydes, such as glutaraldehyde; bis-N-hydroxysuccinimide esters,such as ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester),disuccinimidyl glutarate, disuccinimidyl suberate, and ethyleneglycol-bis(succinimidylsuccinate); diisocyantes, such ashexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyldiglycidyl ether; dicarboxylic acids, such as succinyldisalicylate;3-maleimidopropionic acid N-hydroxysuccinimide ester, and the like.

Other modifications include the addition of one or more amino acids atthe N- or C-terminus, such as histidine residues to allow binding toNi²⁺ derivatized surfaces, or cysteine residues to allow disulfidebridge formation or binding to Sulfolink™ agarose. Alternatively, theantibody may be modified to include one or more chemical spacers at theN-terminus or C-terminus in order to distance the recombinant antibodyoptimally from the solid support. Spacers that can be used include butare not limited to 6-aminohexanoic acid; 1,3-diamino propane;1,3-diamino ethane; and short amino acid sequences, such as polyglycinesequences, of 1 to 5 amino acids.

In an alternative embodiment, the recombinant antibodies optionally canbe conjugated to a carrier protein, such as bovine serum albumin (BSA),casein, or thyroglobulin, in order to immobilize them onto a solidphase.

In another embodiment, the present invention provides for modificationof the recombinant antibodies to incorporate a detectable label.Detectable labels according to the invention preferably are molecules ormoieties which can be detected directly or indirectly and are chosensuch that conjugation of the detectable label to the recombinantantibody preferably does not interfere with the specific binding of theantibody to its target HCV protein. Methods of labeling antibodies arewell-known in the art and include, for example, the use of bifunctionalcross-linkers, such as SAMSA (S-acetylmercaptosuccinic anhydride), tolink the recombinant antibody to the detectable label. Othercross-linking reagents such as are known in the art or which similar tothose described above likewise can be employed.

Detectable labels for use with the recombinant antibodies of the presentinvention include, for example, those that can be directly detected suchas radioisotopes, fluorophores, chemiluminophores, enzymes, colloidalparticles, fluorescent microparticles, and the like. The detectablelabel is either itself detectable or may be reacted with one or moreadditional compounds to generate a detectable product. Thus, one skilledin the art will understand that directly detectable labels of theinvention may require additional components, such as substrates,triggering reagents, light, and the like to enable detection of thelabel. Examples of detectable labels include, but are not limited to,chromogens, radioisotopes such as, e.g., ¹²⁵I, ¹³¹I, ³²P, ³H, ³⁵S and¹⁴C), fluorescent compounds (such as fluorescein, rhodamine, rutheniumtris bipyridyl and lanthanide chelate derivatives), chemiluminescentcompounds (such as, e.g., acridinium and luminol), visible orfluorescent particles, nucleic acids, complexing agents, or catalystssuch as enzymes (such as, e.g., alkaline phosphatase, acid phosphatase,horseradish peroxidase, β-galactosidase, β-lactamase, luciferase). Inthe case of enzyme use, addition of, for example, a chromo-, fluoro-, orlumogenic substrate preferably results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, and Raman spectroscopy are optionallyalso useful.

The present invention also provides for the use of labels that aredetected indirectly. Indirectly detectable labels typically involve theuse of an “affinity pair” i.e. two different molecules, where a firstmember of the pair is coupled to the recombinant antibody of the presentinvention, and the second member of the pair specifically binds to thefirst member. Binding between the two members of the pair is typicallychemical or physical in nature. Examples of such binding pairs include,but are not limited to: antigens and antibodies; avidin/streptavidin andbiotin; haptens and antibodies specific for haptens; complementarynucleotide sequences; enzyme cofactors/substrates and enzymes; and thelike.

4. Preparation of Recombinant Antibodies

The recombinant antibodies of the present invention can compriseantigen-binding domain sequences (for example, the V_(H) and/or V_(L)sequences, or a portion thereof) derived from, for example, a monoclonalantibody produced by a human or non-human animal, such as a rodent,rabbit, canine, feline, bovine, equine, porcine, ape or chicken.Alternatively, antigen-binding domains with the desired binding activitycan be selected through the use of combinatorial libraries expressed inlambda phage, on the surface of bacteriophage, bacteria or yeast, orscreened by display on other biological (for example, retrovirus orpolysome) or non-biological systems using standard techniques (see, forexample, Marks, J. D. et. al., J. Mol. Biol. 222:581-597 (1991); Barbas,C. F. III et. al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992)). Thelibraries can be composed of native antigen-binding domains isolatedfrom an immunized or unimmunized host, synthetic or semi-syntheticantigen-binding domains, or modified antigen-binding domains.

In one embodiment of the present invention, the recombinant antibodiescomprise antigen-binding domains derived from monoclonal antibodies thatbind to the HCV protein of interest. Methods of raising monoclonalantibodies against a desired antigen are well known in the art. Forexample, monoclonal antibodies can be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975). In general inthe hybridoma method, a mouse or other appropriate host animal, such asa hamster or macaque monkey, is immunized by multiple subcutaneous orintraperitoneal injections of antigen and a carrier and/or adjuvant atmultiple sites. Two weeks later, the animals are boosted, and about 7 to14 days later animals are bled and the serum is assayed for anti-antigentiter. Animals can be boosted until titer plateaus.

The splenocytes of the mice are extracted and fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (see, for example, Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986); Galfre etal., Nature, 266:550 (1977)). Suitable myeloma cell lines are known inthe art and include, but are not limited to, murine myeloma lines, suchas those derived from MOP-21 and MC-11 mouse tumors (available from theSalk Institute Cell Distribution Center, San Diego, Calif.), as well asSP-2, SP2/0 and X63-Ag8-653 cells (available from the American TypeCulture Collection (ATCC), Manassas, Va.). Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (see, for example, Kozbor, J. Immunol.,133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)). The hybridoma cells thus prepared can be seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

The hybridoma cells obtained through such a selection are then assayedto identify clones which secrete antibodies capable of binding the HCVantigen used in the initial immunization, for example, byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-immunoassay (EIA or ELISA). The bindingaffinity of the monoclonal antibody can optionally be determined, forexample, by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, the clones may be subcloned by limiting dilutionprocedures, for example the procedure described by Wands et al.(Gastroenterology 80:225-232 (1981)), and grown by standard methods(see, for example, Goding, ibid.). Suitable culture media for thispurpose include, for example, D-MEM, IMDM or RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascitestumors in an animal.

The monoclonal antibodies secreted by the subclones optionally can beisolated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In one embodiment of the present invention, the recombinant antibodiesare derived from monoclonal antibodies raised to a HCV antigen derivedfrom a diagnostically relevant region of an HCV protein. In anotherembodiment, the recombinant antibodies are derived from monoclonalantibodies raised to a HCV antigen derived from a diagnosticallyrelevant region of HCV core, E2, NS3, NS4 or NS5 protein. In a furtherembodiment, the recombinant antibodies are derived from monoclonalantibodies raised to a HCV antigen comprising all or a fragment of: (a)the region of the core protein defined by amino acids 1-150 of the HCVpolyprotein, (b) the region of the NS3 protein defined by amino acids1192 to 1457 of the HCV polyprotein, (c) the regions of the NS4 proteindefined by amino acids 1920 to 1935 or amino acids 1676 to 1931 of theHCV polyprotein, or (d) the region of the NS5 protein defined by aminoacids 2054 to 2995 of the HCV polyprotein. In another embodiment, therecombinant antibodies are derived from monoclonal antibodies raised toa HCV antigen comprising all or a fragment (for example, a fragmentcomprising one or more epitopes) of: (a) the region of the core proteindefined by amino acids 1-50 of the HCV polyprotein, (b) the region ofthe NS3 protein defined by amino acids 1192-1457 of the HCV polyprotein,(c) the region of the NS4 protein defined by amino acids 1696-1931 ofthe HCV polyprotein, and/or (d) the region of the NS5 protein defined byamino acids 1932 to 2191 or amino acids 2188 to 2481. In a furtherembodiment, the recombinant antibodies are derived from monoclonalantibodies raised to a HCV antigen comprising a sequence substantiallyidentical to the sequence as set forth in any one of SEQ ID NOs:33, 34,35, 36, or 37.

In still a further embodiment, the recombinant antibody is derived frommonoclonal antibodies raised to an epitope from the HCV core proteinselected from the group consisting of: (a) an epitope comprised by theamino acid sequence as set forth in SEQ ID NO:100; (b) an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:101; and(c) an epitope comprised by the amino acid sequence as set forth in SEQID NO:102. In yet another embodiment, the recombinant antibody isderived from monoclonal antibodies raised to an epitope from the HCV NS3protein selected from the group consisting of: (a) an epitope comprisedby the amino acid sequence as set forth in SEQ ID NO:103; (b) an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:104; and(c) an epitope comprised by the amino acid sequence as set forth in SEQID NO:105. In still yet another embodiment, the recombinant antibody isderived from monoclonal antibodies raised to an epitope from the HCV NS4protein selected from the group consisting of: (a) an epitope comprisedby the amino acid sequence as set forth in SEQ ID NO:106; (b) an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:107; and(c) an epitope comprised by the amino acid sequence as set forth in SEQID NO:108. And in another embodiment, the recombinant antibody isderived from monoclonal antibodies raised to an epitope from the HCV NS5protein selected from the group consisting of: (a) an epitope comprisedby the amino acid sequence as set forth in SEQ ID NO:109; (b) an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:110; and(c) an epitope comprised by the amino acid sequence as set forth in SEQID NO:111.

Optionally the monoclonal antibody is expressed by a cell line selectedfrom the group consisting of anti-HCV Core 201-603-195, anti-HCV Core14-153-462, anti-HCV NS3 17-903-127, anti-HCV NS4 E99H6C34, and anti-HCVNS5 48-311-387.

Once a monoclonal antibody has been prepared, DNA encoding themonoclonal antibody or the variable regions thereof can readily beisolated by standard techniques, for example by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains or the variable regions of the monoclonalantibody, or by RT-PCR of the mRNA encoding the monoclonal antibodyusing primers to conserved regions (for example, the IgG primer setsavailable from Novagen (EMD Biosciences, Inc.), San Diego, Calif.).

Once isolated, the DNA can be, for example, cloned into an appropriateexpression vector and introduced into a suitable host cell, such as E.coli cells, yeast cells, simian COS cells, Chinese hamster ovary (CHO)cells, human embryonic kidney (HEK) cells (for example, HEK 293), ormyeloma cells that do not otherwise produce immunoglobulin protein, inorder to produce recombinant monoclonal antibodies. Optionally, in oneembodiment, the anti-HCV mouse-human chimeric antibodies of theinvention are produced in a Chinese hamster Ovary (CHO) cell line, whichis advantageous in that they can be produced in quantities sufficientfor commercial use.

Alternatively, the DNA encoding the monoclonal antibody or the variableregions thereof can be used to produce chimeric antibodies, humanizedantibodies and antibody fragments by standard methods known in the art.

For example, chimeric monoclonal antibodies can be produced by cloningthe DNA encoding the variable regions of the monoclonal antibody intomammalian expression vector(s) containing antibody heavy and light chainconstant region genes derived from a different host species. Manyeukaryotic antibody expression vectors that are either stably integratedor exist as extrachromosomal elements have been described and are knownto those of ordinary skill in the art. In general, antibody expressionvectors are plasmids comprising the gene encoding the heavy chainconstant region and/or the gene encoding the light chain constantregion, an upstream enhancer element and a suitable promoter. Forexample, for human constant regions, the vector can comprise the humanIgG1 (human Cγ1) and human kappa constant region (human Cκ) genes andthe immunoglobulin H chain enhancer element. The vector can also containa bacterial origin of replication and selection marker. Optionalinclusion of a selection marker, as is known in the art, allows forselection and amplification under defined growth conditions, for examplethe dihydrofolate reductase (DHFR) gene provides for selection andamplification in mammalian cells with methotrexate. Construction of anappropriate for antibody expression starting from a commercial mammalianexpression vector, can be readily achieved by the skilled technician.Representative examples of recombinant antibody expression vectors areprovided in FIGS. 1, 3, 5, 7 and 12.

Introduction of the expression construct(s) into appropriate host cellsresults in production of complete chimeric antibodies of a definedspecificity (see, for example, Morrison, S. L. et al., Proc. Natl. Acad.Sci. USA 81: 6851-6855 (1984)). The heavy and light chain codingsequences can be introduced into the host cell individually on separateplasmids or together on the same vector.

Depending on the vector system utilized, many different immortalizedcell lines may serve as suitable hosts, these include but are notlimited to myeloma (for example, X63-Ag8.653), hybridoma (for example,Sp2/0-Ag14), lymphoma, insect cells (for example sf9 cells), humanembryonic kidney cells (for example, HEK 293) and Chinese Hamster Ovary(CHO) cells. The expression constructs can be introduced into the hostcells using a variety of techniques known in the art, including but notlimited to, calcium phosphate precipitation, protoplast fusion,lipofection, retrovirus-derived shuttle vectors, and electroporation.

Chimeric antibodies and antibody fragments can also be produced in otherexpression systems including, but not limited to, baculovirus, yeast,bacteria (such as E. coli), and in vitro in cell-free systems such asrabbit reticulocyte lysate.

The recombinant antibody can be isolated from the host cells by standardimmunoglobulin purification procedures such as, for example, cross-flowfiltration, ammonium sulphate precipitation, protein A chromatography,hydroxylapatite chromatography, gel electrophoresis, dialysis, affinitychromatography, or combinations thereof.

Alternatively, antibody fragments can be generated from a purifiedantibody preparation by conventional enzymatic methods, for example,F(ab′)₂ fragments can be produced by pepsin cleavage of the intactantibody, and Fab fragments can be produced by briefly digesting theintact antibody with papain.

Recombinant bispecific and heteroconjugate antibody fragments havingspecificities for at least two different antigens can be prepared asfull length antibodies or as antibody fragments (such as F(ab′)₂bispecific antibody fragments). Antibody fragments having more than twovalencies (for example, trivalent or higher valency antibody fragments)also are contemplated. Bispecific antibodies, heteroconjugateantibodies, and multi-valent antibodies can be prepared by standardmethods known to those skilled in the art.

5. Testing of Recombinant Antibodies

The ability of the recombinant antibody to specifically bind to thetarget HCV antigen can be assessed by standard immunological techniques(see, for example, Current Protocols in Immunology, Coligan, J. E., etal. (ed.), J. Wiley & Sons, New York, N.Y.). For example, byradioimmunoassay (RIA) or enzyme immunoassay (EIA or ELISA). In oneembodiment of the present invention, the recombinant antibodydemonstrates substantially the same specificity as the monoclonalantibody from which the antigen-binding domains are derived.

The recombinant antibodies optionally can also be tested for theirbinding affinity to the target HCV antigen by measuring the equilibriumdissociation constant (K_(D)) by standard techniques. In one embodimentof the present invention, the recombinant antibodies (e.g., chimericantibodies) have a K_(D) less than about 1 μM. In another embodiment,the recombinant antibodies (e.g., chimeric antibodies) have a K_(D) lessthan about 100 nM. In yet another embodiment, optionally the recombinantantibody is chimeric antibody that specifically binds to a HCV proteinepitope, wherein said chimeric antibody is selected from the groupconsisting of:

(a) a HCV core chimeric antibody having an equilibrium dissociationconstant (K_(D)) of from about 0.1 nM to about 1.0 nM, optionally ofabout 0.4 nM, about 0.5, nM, about 0.6 nM, about 0.7 nM, or about 0.8 nM(e.g., as described in Example 9);

(b) a HCV NS3 chimeric antibody having an equilibrium dissociationconstant (K_(D)) of from about 10.0 nM to about 100.0 nM, optionally ofabout 50 nM, about 56 nM, about 62 nM, about 68 nM, or about 74 nM(e.g., as described in Example 10);

(c) a HCV NS4 chimeric antibody having an equilibrium dissociationconstant (K_(D)) of from about 0.1 nM to about 1.0 nM, optionally ofabout 0.3 nM, about 0.4 nM, about 0.5 nM, or about 0.6 nM (e.g., asdescribed in Example 11); and

(d) a HCV NS5 chimeric antibody having an equilibrium dissociationconstant (K_(D)) of from about 1.0 nM to about 10.0 nM, optionally ofabout 8.4 nM, about 8.6 nM, about 8.8 nM, or about 9.0 nM (e.g., asdescribed in Example 12).

Other standard tests also can be done on the antibodies, for example,the pI value of the antibodies can be obtained, such as for the chimericantibodies as described in Example 13. In one embodiment, the chimericantibodies according to the invention which specifically bind to a HCVprotein epitope optionally each comprise a pI value ranging from about7.8 to about 9.0. In another embodiment, the chimeric antibody isselected from the group consisting of: (a) a HCV core chimeric antibodyhaving a pI value of from about 8.8 to about 9.2, optionally about 9.0;(b) a HCV NS3 antibody having a pI value of between about 8.5 and about9.0; (c) a HCV NS4 chimeric antibody having a pI value of between about7.8 and about 8.7, and (d) a HCV NS5 chimeric antibody having a pI valueof between about 8.0 and about 8.9.

Optionally, the recombinant antibodies (e.g., chimeric antibodies) aresubjected to epitope mapping procedures to identify the region of thetarget antigen to which they bind. A variety of methods of epitopemapping are known in the art (see, for example, Current Protocols inImmunology, Coligan, J. E., et al. (ed.), J. Wiley & Sons, New York,N.Y.) and include, for example, phage and yeast display methods. Phageand yeast display methods can also be combined with random mutagenesistechniques in order to more precisely map the residues of the targetantigen involved in antibody binding (see, for example, Chao, G., etal., J. Mol. Biol., 10:539-50 (2004)).

In one embodiment of the present invention, the residues of the targetantigen to which the recombinant antibodies bind are identified by atechnique that combines scanning alanine mutagenesis with yeast display.Non-limiting examples of this procedure are provided in the Examplessection below. The technique generally involves the preparation of aseries of oligonucleotides encoding peptides each representing thetarget region of the antigen and in which each individual amino acid inthis region was sequentially substituted by alanine. The target regionof the antigen is determined either from the antigen used in the initialimmunization to prepare the parent monoclonal antibody, or from apreliminary “low-resolution” screening using yeast or phage display. Awildtype version of the antigen is used as a control. Eacholigonucleotide is cloned into an appropriate yeast display vector andeach alanine mutant transformed into a suitable host, such as E. coli.Plasmid DNA is extracted and sequenced and clones are selected based onsequencing. Yeast display vectors are known in the art and arecommercially available (for example, pYD1 available from Invitrogen,Carlsbad, Calif.).

The selected clones are then transformed into Saccharomyces cerevesiaecells, for example EBY100 cells (Invitrogen Corporation, Carlsbad,Calif.) and individual yeast clones cultured and induced for peptideexpression. The induced yeast cells expressing the alanine mutants onthe cell surface are incubated with the recombinant antibody and boundantibody is detected by conventional methods, for example using alabeled secondary antibody. Key residues in the target antigenic regioncan then be determined based on the identification of alanine mutantsunable to bind to the recombinant antibody. A loss of antibody bindingactivity indicates that the mutant includes an alanine residue at aposition that forms part of the epitope for the recombinant antibody.

6. Uses of Recombinant Antibodies

The recombinant antibodies of the present invention are suitable foruse, for example, as diagnostic reagents for the detection of HCV,and/or as standardization reagents, positive control reagents orcalibrator reagents in assays or kits for the detection of HCVantibodies in place of traditional plasma or serum. Standardizationreagents can be employed, for example, to establish standard curves forinterpolation of antibody concentration. Positive controls can beemployed to establish assay performance characteristics and/orquantitate and monitor the integrity of the antigen(s) used in theassay. The present invention also provides for the use of a plurality ofthe recombinant antibodies, each recombinant antibody capable ofspecifically binding to a different HCV antigen, as standardizedantibody sensitivity panels. Such sensitivity panels can be employed,for example, in place of traditional plasma or serum for quality controlof HCV antibody detection kits, to establish assay performancecharacteristics and/or quantitate and monitor the integrity of theantigen(s) used in the assay. The present invention also contemplatesthe use of the recombinant antibodies in the treatment or prevention ofa HCV infection.

One embodiment of the present invention thus provides for animmunodiagnostic reagent comprising one or more recombinant antibodies,each capable of specifically binding a diagnostically relevant region ofa HCV protein.

In one embodiment of the present invention, the immunodiagnostic reagentcomprises a plurality of (for example, two or more) recombinantantibodies each capable of detecting a different HCV antigen.

The immunodiagnostic reagent can be tailored for a specific end use byappropriate selection of the recombinant antibodies it comprises, thusmaking the immunodiagnostic reagent compatible with a number of existingHCV detection assay formats and kits. Tailoring the immunodiagnosticreagent in this manner also allows the reagent to be optimized fordetection of certain stages of HCV infection. For example, animmunodiagnostic reagent comprising recombinant antibodies to HCV coreand NS3 proteins, and optionally to HCV E2 protein, will allow fordetection of early stage HCV infections, which can be of particularimportance for example in screening blood products. One embodiment ofthe present invention thus provides for an immunodiagnostic reagentcomprising a recombinant antibody specific for HCV core protein, arecombinant antibody specific for HCV NS3 protein, and optionally one ormore of: a recombinant antibody specific for E2 protein, a recombinantantibody specific for HCV NS4 protein and a recombinant antibodyspecific for HCV NS5 protein, which is suitable for detection of earlystage HCV infection and/or for standardization of kits and assays forearly stage HCV infection.

In one embodiment of the present invention, each antibody included inthe immunodiagnostic reagent shows a reactivity to its target antigenthat is greater than or equal to that of a human serum sourced controlunder substantially identical assay conditions.

The present invention further provides for a method of standardizing HCVantibody detection assays using an immunodiagnostic reagent comprising aplurality of recombinant antibodies, each capable of specificallybinding to a different HCV antigenic protein, as a sensitivity panel.

The present invention additionally provides for a method for detectingthe presence of HCV antigens which comprises contacting a test samplesuspected of containing HCV antigens with an immunodiagnostic reagentcomprising one or more recombinant antibodies, each capable ofspecifically binding an HCV antigen, under conditions that allowformation of recombinant antibody:antigen complexes and detecting anyrecombinant antibody:antigen complexes formed.

The present invention also encompasses a method for detecting thepresence of HCV antibodies which comprises contacting a test samplesuspected of containing HCV antibodies with one or more antigensspecific for the HCV antibodies, under conditions that allow formationof antigen/antibody complexes, detecting the antigen:antibody complexes,and employing an immunodiagnostic reagent comprising one or morerecombinant antibodies, each capable of specifically binding one of theantigens employed in the method, as a positive control orstandardization reagent.

The immunodiagnostic reagents of the present invention are suitable foruse with assays and kits monitoring HCV responses in man as well asother vertebrate species susceptible to HCV infection and capable ofgenerating an antibody response thereto. The immunodiagnostic reagentsthus have human medical as well as veterinary applications.

Another aspect of the present invention provides the amino acid andcorresponding nucleic acid sequences of the variable regions ofantibodies specific for HCV core, NS3, NS4 and NS5 proteins,respectively. These sequences have utility in the generation of otherrecombinant antibodies as is known in the art, as well as for theidentification of significantly homologous variable regions of human orhumanized antibodies, such as those contained in a library or bank ofsuch antibodies.

The present invention also encompasses the use of the recombinantantibodies and variable regions described herein in directed molecularevolution technologies such as phage display technologies, and bacterialand yeast cell surface display technologies, in order to produce novelrecombinant antibodies in vitro (see, for example, Johnson et al.,Current Opinion in Structural Biology 3:564 (1993) and Clackson et al.,Nature 352:624 (1991)).

Optionally the immunodiagnostic reagent of the invention, e.g., thechimeric antibodies, can be used in commercial platform immunoassays,e.g., as described in Example 14. For instance, the four chimericantibodies (HCV core, NS3, NS4 and NS5) prepared as described herein canbe used in a variety of Abbott Laboratories HCV blood screening assayson the Prism®, AxSYM®, ARCHITECT® and EIA (Bead) platforms, as well asin other commercial and/or in vitro diagnostic assays as quality controlreagents (e.g., sensitivity panels, calibrators, and positive controls).

7. Compositions Comprising Recombinant Antibodies

As noted above the recombinant antibodies of the present invention canbe used diagnostically and/or therapeutically. One aspect of the presentinvention thus also provides for diagnostic and pharmaceuticalcompositions comprising one or more recombinant antibodies and asuitable carrier, diluent, and/or excipient. Suitable carriers,diluents, and/or excipients are well known in the art. Accordingly, alsoprovided is the use of one or more recombinant antibodies of theinvention for the manufacture of a medicament. For the preparation ofpharmaceutical compositions, the carriers, diluents and excipients willbe pharmaceutically acceptable. If desired, an adjuvant or other activeingredient may be included in the pharmaceutical compositions.

For administration to an animal, the pharmaceutical compositions can beformulated for administration by a variety of routes. For example, thecompositions can be formulated for oral, topical, rectal or parenteraladministration or for administration by inhalation or spray. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrathecal, intrasternal injection or infusiontechniques. As indicated above, such pharmaceutical compositions areused in the treatment of various conditions in animals, includinghumans.

The pharmaceutical compositions preferably comprise a therapeuticallyeffective amount of the recombinant antibody of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For an antibody, the therapeutically effectiveamount can be estimated initially, for example, either in cell cultureassays or in animal models, usually in rodents, rabbits, dogs, pigs orprimates. The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inthe animal to be treated, including humans.

Various diagnostic compositions and pharmaceutical compositions suitablefor different routes of administration and methods of preparingpharmaceutical compositions are known in the art and are described, forexample, in “Remington: The Science and Practice of Pharmacy” (formerly“Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams& Wilkins, Philadelphia, Pa. (2000).

8. Kits Comprising Recombinant Antibodies

The present invention further provides for therapeutic, diagnostic andquality control kits comprising one or more recombinant antibodies ofthe invention.

a. Diagnostic and Quality Control Kits

One aspect of the present invention provides diagnostic kits for thedetection of HCV. The kits comprise one or more recombinant antibodiesof the present invention. The recombinant antibodies can be provided inthe kit as detection reagents, either for use to capture and/or detectHCV antigens or for use as secondary antibodies for the detection ofantigen:antibody complexes. Alternatively, the recombinant antibodiescan be provided in the kit as a positive control reagent, astandardization reagent, calibration reagent or a sensitivity panel. Invarious embodiments, the diagnostic kit can further comprise reagentsfor detection of HCV antigens or reagents for the detection of HCVantibodies. In one embodiment, the present invention provides adiagnostic kit comprising reagents for detection of HCV antibodies,including one or more antigens specific for the HCV antibodies, and apositive control or standardization reagent comprising one or morerecombinant antibodies of the invention, each capable of specificallybinding one of the one or more antigens included in the kit.

As discussed above, optionally the immunodiagnostic reagent of theinvention, e.g., the chimeric antibodies, can be used in kits forcommercial platform immunoassays (e.g., HCV blood screening assays onthe Prism®, AxSYM®, ARCHITECT® and EIA (Bead) platforms, as well as inother commercial and/or in vitro diagnostic assay) as quality controlreagents (e.g., sensitivity panels, calibrators, and positive controls).Preparation of quality control reagents is well known in the art, and isdescribed, e.g., on a variety of immunodiagnostic product insert sheets.HCV sensitivity panel members optionally can be prepared in varyingamounts containing known quantities of HCV antibody ranging from “low”to “high”, e.g., by spiking known quantities of chimeric antibodiesaccording to the invention into an appropriate assay buffer (e.g., aphosphate buffer). These sensitivity panel members optionally are usedto establish assay performance characteristics, and further optionallyare useful indicators of the integrity of the immunoassay kit reagents,and the standardization of assays.

In another embodiment, the present invention provides for a qualitycontrol kit comprising one or more recombinant antibodies of the presentinvention for use as a sensitivity panel to evaluate assay performancecharacteristics and/or quantitate and monitor the integrity of theantigen(s) used in the assay.

The recombinant antibodies provided in the kit can incorporate adetectable label, such as a fluorophore, radioactive moiety, enzyme,biotin/avidin label, chromophore, chemiluminescent label, or the like,or the kit may include reagents for labeling the recombinant antibodiesor reagents for detecting the recombinant antibodies. The recombinantantibodies can be provided in separate containers or pre-dispensed intoan appropriate assay format, for example, into microtiter plates.

The kits can optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample, may also beincluded in the kit. The kit may additionally include one or morecontrols. One or more of the components of the kit may be lyophilizedand the kit may further comprise reagents suitable for thereconstitution of the lyophilized components.

The various components of the kit are provided in suitable containers.As indicated above, one or more of the containers may be a microtiterplate. Where appropriate, the kit may also optionally contain reactionvessels, mixing vessels and other components that facilitate thepreparation of reagents or the test sample. The kit may also include oneor more instrument for assisting with obtaining a test sample, such as asyringe, pipette, forceps, measured spoon, or the like.

The kit can optionally include instructions for use, which may beprovided in paper form or in computer-readable form, such as a disc, CD,DVD or the like.

b. Therapeutic Kits

The present invention additionally provides for therapeutic kits orpacks containing one or more of recombinant antibodies of the inventionor a pharmaceutical composition comprising one or more of therecombinant antibodies for use in the treatment or prevention of a HCVinfection. Individual components of the kit can be packaged in separatecontainers, associated with which, when applicable, can be a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency of manufacture, use or sale for human oranimal administration. The kit can optionally further contain one ormore other therapeutic agents for use in combination with therecombinant antibodies of the invention. The kit may optionally containinstructions or directions outlining the method of use or dosing regimenfor the recombinant antibodies and/or additional therapeutic agents.

When one or more components of the kit are provided as solutions, forexample an aqueous solution, or a sterile aqueous solution, thecontainer means may itself be an inhalant, syringe, pipette, eyedropper, or other such like apparatus, from which the solution may beadministered to a subject or applied to and mixed with the othercomponents of the kit.

The components of the kit may also be provided in dried or lyophilizedform and the kit can additionally contain a suitable solvent forreconstitution of the lyophilized components. Irrespective of the numberor type of containers, the kits of the invention also may comprise aninstrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, syringe, pipette,forceps, measured spoon, eye dropper or similar medically approveddelivery vehicle.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It will be understood that theseexamples are intended to describe illustrative embodiments of theinvention and are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 Preparation of a Human-Mouse Chimeric AntibodySpecific for Hepatitis C Virus Core Protein #1 Preparation of aHybridoma Cell Line

The hybridoma cell line anti-HCV Core 201-603-195 was developed usingthe PEG-mediated fusion technique described in Galfre et al., Nature,266:550 (1977). Briefly, BALB/c female mice were immunized with apurified HCV recombinant core antigen known as pλ core (corresponding toamino acids 1-50 of the HCV polyprotein). The animal boost regimenutilized the Freunds Adjuvant System and sera samples were monitored inan HCV enzyme immunoassay (EIA) until an anti-HCV titer was identified.For the EIA, a HCV recombinant core antigen comprising core amino acids1-150 (as well as NS3 amino acids 1192-1457) were coated on 96 well EIAplates for at least 1 hour at room temperature, and then were blockedwith 2% BSA/PBS buffer for 1 hour. The mouse sera samples were addedinto the coated wells and the plates incubated for at least 1 hour atroom temperature. After incubation, the plates were washed and incubatedwith horseradish peroxidase (HRP) labeled goat-anti mouse IgG antibodyfor about 1 hour. The plates were developed usingO-Phenylenediamine-2HCl (OPD) and read at an optical density of 492 nm.

The mouse spleen cells were then fused with the SP2/0 myeloma andcultured at 37° C. in HAT-supplemented Iscove's Modified Dulbecco'sMedium (IMDM) containing 10% fetal bovine serum (GIBCO). The hybridomaswere tested 10-14 days later for anti-HCV reactivity by EIA. Hybridomassecreting anti-HCV monoclonal antibodies were cloned by standard singlecell dilution techniques and subsequent clones were tested forreactivity by EIA. Positive cultures were expanded in IMDM containing10% FBS and then frozen back in a cryopreservative for long term storagein liquid nitrogen.

The hybridoma cell line anti-HCV Core 201-603-195 was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on Nov. 21, 2006 under Accession No. PTA-8027.

Isolation of mRNA and Identification of Mouse V_(H) and V_(L) Sequences

The anti-HCV Core 201-603-195 hybridoma cell line obtained as describedabove was cultured in Hybridoma Serum-Free Medium (HSFM) to obtain˜5×10⁶ cells for mRNA purification. Poly A+ mRNA was isolated from thecells and purified using Oligotex Direct mRNA Micro Kit (QIAGEN Inc.,Valencia, Calif.) following the manufacturer's instructions.

The purified mRNA was then used as a template in a RT-PCR reaction usingthe Mouse Ig-Primer® set (EMD Biosciences Inc. (Novagen), San Diego,Calif.). Each Mu Ig V_(H) 5′-A Primer, Mu Ig V_(H) 5′-B Primer, Mu IgV_(H) 5′-C Primer, Mu Ig V_(H) 5′-D Primer, Mu Ig V_(H) 5′-E Primer, andMu Ig V_(H) 5′-F Primer was individually paired with Mu IgG V_(H) 3′-2Primer for the V_(H) RT-PCR reaction. Each Mu Ig kappa V_(L) 5′-APrimer, Mu Ig kappa V_(L) 5′-B Primer, Mu Ig kappa V_(L) 5′-C Primer, MuIg kappa V_(L) 5′-D Primer, Mu Ig kappa V_(L) 5′-E Primer, Mu Ig kappaV_(L) 5′-F Primer, and Mu Ig kappa V_(L) 5′-G Primer was individuallypaired with Mu Ig kappa V_(L) 3′-1 Primer for the V_(L) RT-PCR reaction.Each RT-PCR reaction was executed in 2× reaction Buffer (dNTP), each 5′and 3′ primer pair and 2.5 units of RT-Platinum Taq HiFi Mix (InvitrogenCorp., Carlsbad, Calif.). The cDNA synthesis and pre-denaturation wereperformed at 1 cycle of 50° C. for 30 minutes and 1 cycle of 94° C. for2 minutes, followed by the PCR reaction comprising denaturation for 1minute at 94° C.; annealing 1 minute at 50° C. and extension 2 minutesat 68° C., with a final extension 6 minutes at 72° C. A total of 45cycles were performed.

Positive PCR products were observed from the heavy chain (H) primer setsof B and C (VH-B, VH-C) and from the light chain (L) primer sets of A,B, E, F, G (VL-A, VL-B, VL-E, VL-F, VL-G). All positive PCR productswere gel purified and cloned into the pCR TOPO 2.1 TA vector (InvitrogenCorp., Carlsbad, Calif.) and transformed into E. coli DH5α. The plasmidDNA was extracted from the E. coli cells using the QIAprep spin miniprepKit (QIAGEN Inc., Valencia, Calif.) following the manufacturer'sinstructions and the V_(H) or V_(L) insert confirmed by EcoR I digestionfor each PCR product. The final TA clone for each of the V_(H) or V_(L)inserts was selected by sequence alignment for either the light chainvariable or the heavy chain variable using Vector NTI Advance™ software(Invitrogen Corp., Carlsbad, Calif.). In brief, each TA clone was grownin LB broth overnight with shaking at 37° C. Plasmid DNA from each clonewas purified using the QIAprep spin miniprep kit (QIAGEN) and sequencedusing M13 forward and reverse primers and the Big Dye Terminator v3.1cycle sequencing kit (Applied Biosystems, Foster City, Calif.).Sequences were inputted into the Vector NTI Advance software andaligned. Sequences which aligned completely, i.e. had no mutations,insertions or deletions, were considered correct clones and wereselected for further development. The sequence results identified VL-GTA clone number 1 as containing the correct V_(L) sequence (SEQ ID NO:8,which encodes the polypeptide shown in SEQ ID NO:2) from the hybridomacell line and the VH-C TA clone number 1 as containing the correct V_(H)sequence (SEQ ID NO:7, which encodes the polypeptide shown in SEQ IDNO:1) (see FIGS. 2A-D).

Cloning the V_(H) and V_(L) Genes into pBOS Vectors

The pBOS vectors are known in the art and are described, e.g., in US2005/0227289 (incorporated by reference for its teachings regarding theuse of these vectors and the vectors themselves).

Using the VL-G TA clone number 1 as the template, a pair of PCR primerswere designed to clone out the mouse V_(L) sequence, namely, a HCV CoreAntibody V_(L) 5′-end primer comprising the sequence

[SEQ ID NO: 3] 5′-GCTCGCGATGCGACATTGTGATGTCACAGTCT-3′, 

and a HCV Core Antibody V_(L) 3′-end primer comprising the sequence

[SEQ ID NO: 4] 5′-CACCGTACGTTTTATTTCCAGCTTGGT-3′.

The 5′-end primer (SEQ ID NO:3) contained a partial Kappa signalsequence and a Nru I restriction site, and the 3′-end primer (SEQ IDNO:4) contained a BsiW I restriction site. In addition, using the VH-CTA clone number 1 as the template, a pair of primers was designed toclone out the mouse V_(H) sequence, namely, a HCV Core Antibody V_(H)5′-end primer comprising the sequence

[SEQ ID NO: 5] 5′-TTGTCGCGATTTTAAAAGGTGTCCAGTGCCAGATCCAGTTGGTGCAGTCTGGACCT-3′,

and a HCV Core Antibody V_(H) 3′-end primer comprising the sequence

[SEQ ID NO: 6] 5′-TTGGTCGACGCTGAGGAGACGGTGACTGAGGTT-3′.

For this pair of primers, the 5′-end primer (SEQ ID NO:5) contained apartial heavy chain signal sequence and an Nru I restriction site andthe 3′-end primer (SEQ ID NO:6) contained a Sal I restriction site.

The PCR was executed in 2×Pfx amplification buffer using 15 pmol each ofthe 5′-end and 3′-end primers, 1.25 units of Pfx DNA polymerase(Invitrogen, Carlsbad, Calif.), and 100 ng of TA clone plasmid DNA. ThePCR was performed for 30 cycles of 15 seconds at 94° C. followed by 1minute at 68° C.

The V_(L) and V_(H) sequences were independently amplified by PCR usingthe above primer pairs and the VL-G TA clone number 1 and VH-C TA clonenumber 1, respectively, as the templates. The V_(L) and V_(H) PCRproducts were restriction enzyme trimmed by Nru I/BsiW I and Nru I/Sal Idigestion, respectively, and then cloned into either the pBOS-hck vector(for the V_(L) sequence) or the pBOS-hcg vector (for the V_(H) sequence)and transformed into E. coli DH5α. The pBOS-hck vector comprises theampicillin resistance gene, pUC origin, SV40 origin, EF-1a promoter,kappa signal peptide and human kappa gene (hck). The pBOS-hcg vectorcomprises the ampicillin resistance gene, pUC origin, SV40 origin, EF-1apromoter, heavy chain signal peptide and human constant IgG gene(hcg1,2,non-a).

The transformed E. coli clones were grown in LB broth overnight withshaking at 37° C. Plasmid DNA was purified from each individual clonewith the QIAprep spin miniprep kit (QIAGEN) followed by sequencing usingthe Bigdye Terminator v3.1 cycle sequencing kit (Applied Biosystems).Plasmids pBOS 201-603-195-L-T9 (containing the V_(L) sequence) and pBOS201-603-195-H-T4 (containing the V_(H) sequence) were selected bysequencing. Once the respective pBOS clones were identified, separate E.coli DH5α cell banks containing either the pBOS 201-603-195-L-T9 plasmidor the pBOS 201-603-195-H-T4 plasmid were made to preserve the pBOSclones.

Chimeric Antibody Functional Testing

pBOS 201-603-195-L-T9 and pBOS 201-603-195-H-T4 plasmid DNA was preparedusing the Endofree plasmid Maxi-kit (QIAGEN, CA) by standard techniques.The high purity plasmid DNA thus obtained was then transientlytransfected into COS 7L cells by electroporation (Gene Pulser, BIO-RAD).The transfected COS 7L cells were incubated at 37° C. in a 5% CO₂incubator for three days, then harvested by centrifugation at 4000 rpmfor 20 minutes and the supernatant collected. Following filtration andstandard protein A agarose (Invitrogen) affinity purification, thepurified anti-HCV core chimeric antibody was assayed by enzymeimmunoassay (EIA) to confirm reactivity with HCV core antigen. The EIAwas conducted as described above using horseradish peroxidase labeledgoat anti-human (“GAH”) IgG antibody as the detecting antibody andvarying concentrations of the chimeric antibody. The EIA resultsdemonstrated that the HCV core chimeric antibody from transientexpression was reactive to the HCV core antigen.

Cloning the V_(H) and V_(L) Sequences into a Stable Expression Vector

The pBOS 201-603-195-L-T9 and pBOS 201-603-195-H-T4 clones were used toconstruct a plasmid clone for stable cell line transfection. First, theSrf I and Not I restriction sites flanking the V_(H) and V_(L) sequencesin the respective vectors were employed to excise each of the V_(H) andV_(L) sequences from the vectors. The excised sequences were gelpurified and then cloned into either the pBV vector (for the V_(H)sequence) or the pJV vector (for the V_(L) sequence). The pJV stufferplasmid comprises a SV40 promoter, a murine DHFR gene, a CMV enhancer,an adenovirus major late (AML) promoter and a lambda stuffer. The pBVstuffer plasmid comprises a CMV enhancer, an adenovirus major late (AML)promoter and a lambda stuffer.

The pBV and pJV clones were screened by Srf I/Not I restriction enzymedigestion to identify those clones containing the correct insert. Oncecorrect pBV and pJV clones had been identified, the heavy chain gene(from pBV) and light chain gene (from pJV) were cloned into one plasmid,by digesting each plasmid with Asc I and Pac I. For the light chain geneplasmid (pJV), Pac I/Asc I digestion produced two distinct bands at 4.8Kb and 1.5 kb. For the heavy chain gene plasmid (pBV), Pac I/Asc Idigestion produced two distinct band at 4.6 kb and 1.2 kb. The 4.8 kbDNA fragment from pJV plasmid and the 4.6 kb DNA fragment from pBV weregel purified and ligated together to form a pBJ plasmid that containedboth the V_(H) and V_(L) sequences. After transformation, the final pBJclone pBJ HCV core 201-603-195 was selected based on screening by SrfI/Not I digestion to confirm that it contained both the V_(H) and V_(L)sequences. The plasmid map for the final pBJ clone (pBJ HCV core201-603-195) for the HCV core chimeric antibody thus obtained is shownin FIG. 1. The V_(H) and V_(L) gene sequences are shown in FIGS. 2A-B(SEQ ID NOs: 7 and 8, respectively). E. coli cell banks containing thepBJ HCV core 201-603-195 clone were made.

Establishing a Stable CHO Cell Line and Expression of the HCV CoreChimeric Antibody

A Chinese Hamster Ovary (CHO, B3.2) cell line that lacks thedihydrofolate reductase (DHFR) gene was used for transfection and stablechimeric antibody expression as described below. The CHO cells werecultured and transfected by standard calcium phosphate mediatedtransfection with the pBJ HCV core 201-603-195 plasmid. The transfectedHCV core CHO cells were cultured for several weeks prior to a singlecell cloning into 96-well plates using the BD FACSAria flow cytometersorter. Once the CHO clones had grown to more than 50% confluency, thesupernatant was tested in an antigen specific EIA to rank theperformance of the CHO clones. The EIA was conducted as described aboveusing varying concentrations of the chimeric antibody. The 16 CHO clonesthat gave the highest signal in the EIA were expanded and re-assayed.Ten CHO clones were then selected based on the highest signal given inthe EIA assay for weaning into CD CHO serum free tissue culture medium,including the clone CHO 201-603-486. Following one more single cellsubcloning using the flow cytometer, HCV core CHO clone 201-603-486-333was expanded for production of chimeric antibody and for development ofcell banks for long-term storage in liquid nitrogen. The chimericantibody was purified from this cell line using standard Protein A(POROS A50, Applied Biosystems) purification procedures followed bySephadex G-25 Superfine desalting column purification.

The HCV core CHO 201-603-486-333 cell line was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on May 2, 2006 under Patent Deposit Designation PTA-7570.

Example 2 Preparation of a Human-Mouse Chimeric Antibody Specific forHepatitis C Virus NS3 Protein Preparation of a Hybridoma Cell Line

The hybridoma cell line anti-HCV NS3 17-903-127 was developed asdescribed in Example 1 using the purified HCV recombinant NS3 antigenknown as CKS-33C-BCD in place of the core antigen. The CKS-33C-BCDrecombinant antigen corresponds to amino acids 1192-1457+1676-1931 ofthe HCV polyprotein. The animal immunization regimen utilized one 200 μgboost in the Freunds Adjuvant System, with a pre-fusion boost in saline2 weeks later.

The hybridoma cell line anti-HCV NS3 17-903-127 was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on Nov. 21, 2006 under Accession No. PTA-8023.

Isolation of mRNA and Identification of Mouse V_(H) and V_(L) Sequences

mRNA was isolated from the hybridoma cell line anti-HCV NS3 17-903-127and the mouse V_(H) and V_(L) sequences obtained by RT-PCR as describedin Example 1 with respect to the HCV Core 201-603-195 hybridoma cellline. Positive PCR products were observed from the heavy chain (H)primer sets of A and F (VH-A, VH-F) and from the light chain (L) primersets of B, C and G (VL-B, VL-C, VL-G). All positive PCR products weregel purified, cloned into the pCR TOPO 2.1 TA vector and transformedinto E. coli. Colony PCR reactions were used to confirm the cloneinsert. Briefly, the PCR was conducted using M13 forward and M13 reverseprimer (Invitrogen), Taq DNA polymerase and boiled E. coli colony as thetemplate. The PCR comprised denaturation 1 minute at 94° C.; annealing 1minute at 50° C. and extension 2 minutes at 72° C., with a finalextension 6 minutes at 72° C. for a total of 30 cycles. The clonesgiving the correct size for the PCR products were grown in LB broth.Each plasmid DNA was purified using QIAprep spin miniprep Kit (QIAGEN)and sequenced using M13 forward and reverse primers with the Big DyeTerminator v3.1 cycle sequencing kit (Applied Biosystems).

The final TA clone was selected by sequence alignment as described inExample 1. The sequence results confirmed that the VL-G clone containedthe correct V_(L) sequence (SEQ ID NO:16 encoding the polypeptide shownin SEQ ID NO:10), while the VH-A and F clones contained the correctV_(H) sequence (SEQ ID NO:15 encoding the polypeptide shown in SEQ IDNO:9) (see also FIGS. 4A-D).

Cloning the V_(H) and V_(L) Genes into pBOS Vectors

Using the VL-G TA clone number 5 as the template, a pair of PCR primerswere designed to clone out the mouse V_(L) sequence, namely a HCV NS3Antibody V_(L) 5′-end primer comprising the sequence

5′-CTGTGGTTCCCCGGCTCGCGATGCGATGTTGTGATGGCCCAAAC

TCCACTCTCCCC-3′[SEQ ID NO:11], and a HCV NS3 Antibody V_(L) 3′-endprimer comprising the sequence

5′-GCGCATGCGTCGTACGTTTTATTTCCAGCTTGGTCCCCCC-3′ [SEQ ID NO:12].

The 5′-end primer contained a partial Kappa signal sequence and a Nru Irestriction site and the 3′-end primer contained a BsiW I restrictionsite. In addition, using the VH TA clone number 2 as the template, apair of primers was designed to clone out the mouse V_(H) sequence,namely, a HCV NS3 Antibody V_(H) 5′-end primer comprising the sequence

5′-GGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGCGAAGTG

AAGCTGGTGGAGTCTG GGGGAGGC-3′ [SEQ ID NO:13], and a HCV NS3

Antibody V_(H) 3′-end primer comprising the sequence

-   -   5′-GCGCATGCATGCATTGTCGACGCGAGGAGACTGTGAGAGTGGTGCCTTGGCCC-3′ [SEQ        ID NO:14].

For this pair of primers, the 5′-end primer contained a partial heavychain signal sequence and an Nru I restriction site and the 3′-endprimer contained a Sal I restriction site.

The V_(L) and V_(H) sequences were independently amplified by PCR usingthe above primer pairs and the VL-G TA clone number 5 and VH TA clonenumber 2, respectively, as the templates. The PCR was executed in 1×PCRamplification Buffer using 20 pmol each of the 5′-end and 3′-endprimers, 1 unit of Taq DNA polymerase (Invitrogen), and 2 μl ofbacterial colony as the template. The PCR was performed employing 30cycles of 30 seconds at 94° C. and 30 seconds at 55° C. followed by 1minute at 72° C.

The V_(L) and V_(H) PCR products were restriction enzyme trimmed by NruI/BsiW I and Nru I/Sal I digestion, respectively, and then cloned intoeither the pBOS-hck vector (for the V_(L) sequence) or the pBOS-hcgvector (for the V_(H) sequence), as described in Example 1. PlasmidspBOS 17-903-127-L (containing the V_(L) sequence) and pBOS 17-903-127-H(containing the V_(H) sequence) were selected by sequencing as describedin Example 1. Once the respective pBOS clones were identified, separateE. coli DH5α cell banks containing either the pBOS 17-903-127-L-T3plasmid or the pBOS 17-903-127-H-T2 plasmid were made to preserve thepBOS clones.

Chimeric Antibody Functional Testing

pBOS 17-903-127-L-T3 and pBOS 17-903-127-H-T2 plasmid DNA was preparedand transiently transfected into COS 7L cells as described in Example 1.The anti-HCV NS3 chimeric antibody was prepared from the transfected COS7L cells as described in Example 1 and assayed by EIA to confirmreactivity with HCV NS3 antigen. The EIA was conducted as described inExample 1 using CKS-33C-BCD HCV NS3 antigen in place of the coreantigen. The EIA results demonstrated that the chimeric antibody fromtransient expression was reactive to the HCV NS3 antigen.

Cloning the V_(H) and V_(L) Sequences into a Stable Expression Vector

The pBOS 17-903-127-L-T3 and pBOS 17-903-127-H-T2 clones were used toconstruct a plasmid clone for stable cell line transfection by firstcloning the sequences into the pBV vector (for the V_(H) sequence) orthe pJV vector (for the V_(L) sequence) and subsequently ligatingtogether the vector fragments obtained from Pac I/Asc I restrictionenzyme digestion as described in Example 1 to provide the pBJ plasmidpBJ HCV NS3 17-903-127 that contains both the V_(H) and the V_(L)sequences. The pBJ clone was screened by Srf I/Not I digestion toconfirm that it contains both sequences. The plasmid map for the finalpBJ clone (pBJ HCV NS3 17-903-127) for the HCV NS3 chimeric antibody isshown in FIG. 3. The V_(H) and V_(L) gene sequences are shown in FIG. 4(SEQ ID NO:15 and SEQ ID NO:16, respectively). E. coli DH5α cell bankscontaining the HCV pBJ HCV NS3 17-903-127 clone were made.

Establishing a Stable CHO Cell Line and Expression of the HCV ChimericAntibody

The pBJ HCV NS3 17-903-127 plasmid was transfected by calcium phosphatemediated transfection into the DHFR-deficient Chinese Hamster Ovary cellline as described in Example 1. Culture and single cell cloning wasconducted as described in Example 1. The 96 well plates were tested byantigen specific EIA and clone number 132 was selected for furtherdevelopment. The EIA was conducted as described above. The HCV NS317-903-132 CHO cell clone was cultured and single cell sorted into96-well plates using the BD FACSAria flow cytometer sorter. CHO subclonenumber 171 was selected for weaning into CD-CHO serum free medium(Invitrogen). The final CHO cell line (HCV NS3 17-903-132sc171) wasexpanded for production of the chimeric antibody and for development ofa discovery cell bank for storage in liquid nitrogen. The chimericantibody was purified from this cell line using standard Protein Apurification procedures as described in Example 1.

The HCV NS3 CHO 17-903-132sc171 cell line was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on May 2, 2006 under Patent Deposit Designation PTA-7573.

Example 3 Preparation of a Human-Mouse Chimeric Antibody Specific forHepatitis C Virus NS4 Protein Preparation of a Hybridoma Cell Line

The hybridoma cell line anti-HCV NS4 E99H6C34 was developed as describedin Example 1 using the purified HCV recombinant NS4 antigen known asCKS-C100 in place of the core antigen. The CKS-C100 recombinant antigencorresponds to amino acids 1676-1931 of the HCV polyprotein. The animalboost regimen used the Freunds Adjuvant System and sera samples weremonitored in an HCV EIA until an anti-HCV titer was identified. The E1Awas conducted as described in Example 1 using the CKS-C100 HCV NS4antigen in place of the core antigen. The mouse spleen cells were fusedwith the SP2/0 myeloma and cultured at 37° C. in HAT-supplementedDulbecco's Modified Eagle's Medium (DMEM) containing 20% fetal bovineserum (GIBCO). The hybridomas were tested 10-14 days later for anti-HCVreactivity in an EIA. Hybridomas secreting anti-HCV monoclonalantibodies were cloned by single cell dilution techniques and subsequentclones were tested for reactivity in an EIA. Positive cultures wereexpanded in DMEM containing 10% FBS and frozen back in acryopreservative for long term storage in liquid nitrogen.

The hybridoma cell line anti-HCV NS4 E99H6C34 was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on Nov. 21, 2006 under Accession No. PTA-8021.

Isolation of mRNA and Identification of Mouse V_(H) and V_(L) Sequences

mRNA was isolated from the hybridoma cell line anti-HCV NS4 E99H6C34 andthe mouse V_(H) and V_(L) sequences obtained by RT-PCR as described inExample 1 with respect to the HCV Core 201-603-195 hybridoma cell line.Positive PCR products were observed from the heavy chain (H) primer setof C (VH-C) and from the light chain (L) primer sets of B, C and G(VL-B, VL-C, VL-G). All positive PCR products were gel purified, clonedinto the pCR TOPO 2.1 TA vector and transformed into E. coli DH5α. Theplasmid DNA was extracted from the E. coli cells and the V_(H) or V_(L)insert confirmed by EcoR I digestion for each PCR product. The final TAclone was selected by sequence alignment as described in Example 1. Thesequence results confirmed that VL-G TA clone number 5 contained thecorrect V_(L) sequence (SEQ ID NO:24 encoding a polypeptide as shown inSEQ ID NO:18), while the VH-C TA clone number 16 contained the correctV_(H) sequence (SEQ ID NO:23 encoding a polypeptide as shown in SEQ IDNO:17) (see also FIGS. 6A-D).

Cloning the V_(H) and V_(L) Sequences into pBOS Vectors

Using the VL-G TA clone number 5 as the template, a pair of PCR primerswere designed to clone out the mouse V_(L) sequence, namely, a HCV NS4Antibody V_(L) 5′-end primer comprising the sequence

5′-GCTCGCGATGCGATGTTGTGATGACCCAAAC-3′ [SEQ ID NO:19], and a HCV NS4Antibody V_(L) 3′-end primer comprising the sequence

5′-CACCGTACGTTTGATTTCCAGCTTGGTGC-3′ [SEQ ID NO:20].

The 5′-end primer contained a partial Kappa signal sequence and a Nru Irestriction site and the 3′-end primer contained a BsiW I restrictionsite. In addition, using the VH-C TA clone number 16 as the template, apair of primers was designed to clone out the mouse V_(H) sequence,namely, a HCV NS4 Antibody V_(H) 5′-end primer comprising the sequence

5′-TACTTCGCGACAGATCCAGTTGGTGCAGTC-3′ [SEQ ID NO:21], and a HCV NS4Antibody V_(H) 3′-end primer comprising the sequence

5′-TGGTCGACGCTGAGGAGACTGTGAGAGTGGT-3′ [SEQ ID NO:22].

For this pair of primers, the 5′-end primer contained a partial heavychain signal sequence and a Nru I restriction site and the 3′-end primercontained a Sal I restriction site.

The V_(L) and V_(H) sequences were independently amplified by PCR usingthe above primer pairs and the VL-G TA clone number 5 and VH-C TA clonenumber 16, respectively, as the templates. The PCR was executed in 2×Pfxamplification Buffer using 15 pmol each of the 5′-end and 3′-endprimers, 1.25 units of Pfx DNA polymerase (Invitrogen) and 100 ng of TAclone plasmid DNA as the template. The PCR was performed using 30 cyclesof 15 seconds at 94° C. followed by 1 minute at 68° C.

The V_(L) and V_(H) PCR products were restriction enzyme trimmed by NruI/BsiW I and Nm I/Sal I digestion, respectively, and then cloned intoeither the pBOS-hck vector (for the V_(L) sequence) or the pBOS-hcgvector (for the V_(H) sequence), as described in Example 1. PlasmidspBOS E99H6C34-L-T4 (containing the V_(L) sequence) and pBOSE99H6C34-H-T2 (containing the V_(H) sequence) were selected bysequencing. Once the respective pBOS clones were identified, separate E.coli DH5α cell banks containing either the pBOS E99H6C34-L-T4 plasmid orthe pBOS E99H6C34-H-T2 plasmid were made to preserve the pBOS clones.

Chimeric Antibody Functional Testing

pBOS E99H6C34-L-T4 and pBOS E99H6C34-H-T2 plasmid DNA was prepared andtransiently transfected into COS 7L cells as described in Example 1. Theanti-HCV NS3 chimeric antibody was prepared from the transfected COS 7Lcells as described in Example 1 and assayed by EIA to confirm reactivitywith HCV NS4 antigen (C100 Ag). The EIA was conducted as describedabove. The EIA results demonstrated that the chimeric antibody fromtransient expression was reactive to the HCV NS4 antigen.

Cloning the V_(H) and V_(L) Sequences into a Stable Expression Vector

The pBOS E99H6C34-L-T4 and pBOS E99H6C34-H-T2 clones were used toconstruct a plasmid clone for stable cell line transfection by firstcloning the sequences into the pBV vector (for the V_(H) sequence) orthe pJV vector (for the V_(L) sequence) and subsequently ligatingtogether the vector fragments obtained from Pac I/Asc I restrictionenzyme digestion as described in Example 1 to provide the pBJ plasmidpBJ HCV NS4 E99H6C34 that contains both the V_(H) and the V_(L)sequences. The pBJ clone was screened by Srf I/Not I digestion toconfirm that it contains both sequences. The plasmid map for the finalpBJ clone (pBJ HCV NS4 E99H6C34) for the HCV NS4 chimeric antibody isshown in FIG. 5. The V_(H) and V_(L) gene sequences are shown in FIGS.6A-B (SEQ ID NO:23 and SEQ ID NO:24, respectively). E. coli DH5α cellbanks containing the pBJ HCV NS4 E99H6C34 clone were made.

Establishing a Stable CHO Cell Line and Expression of the HCV ChimericAntibody

The pBJ HCV NS4 E99H6C34 plasmid was transfected by calcium phosphatemediated transfection into the DHFR-deficient Chinese Hamster Ovary cellline as described in Example 1. Culture and single cell cloning wasconducted as described in Example 1. When the CHO clones had grown tomore than 50% confluency, the supernatant was tested in an antigenspecific EIA to rank the performance of the CHO clones. The EIA wasconducted as described above. The 16 CHO clones showing the highestsignal were expanded and re-assayed by an antigen specific EIA. Six CHOclones were selected for weaning into CD CHO serum free tissue culturemedium. HCV NS4 CHO clone #203 was expanded to produce purified chimericantibody and for development of a discovery cell bank for storage inliquid nitrogen. The chimeric antibody was purified from this cell lineusing standard Protein A purification procedures as described in Example1.

The HCV NS4 CHO E99H6C34sc203 cell line was deposited with the AmericanType Culture Collection (ATCC) at 10801 University Boulevard, Manassas,Va. on May 2, 2006 under Patent Deposit Designation PTA-7569.

Example 4 Preparation of a Human-Mouse Chimeric Antibody Specific forHepatitis C Virus NS5 Protein Preparation of a Hybridoma Cell Line

The hybridoma cell line anti-HCV NS5 48-311-387 was developed asdescribed in Example 1 using the HCV recombinant antigen known as CKSEF. The CKS-EF recombinant antigen corresponds to amino acids1932-2191+2188-2481 of the HCV polyprotein. BALB/c female mice wereimmunized once with 200 μg of purified HCV recombinant antigen (CKS-EF)using the Freunds Adjuvant System. Three mice were administered apre-fusion boost 3 days prior to harvesting the spleens.

The hybridoma cell line anti-HCV NS5 48-311-387 was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on Nov. 21, 2006 under Accession No. PTA-8022.

Isolation of mRNA and Identification of Mouse V_(H) and V_(L) Sequences

mRNA was isolated from the hybridoma cell line anti-HCV NS5 48-311-387and the mouse V_(H) and V_(L) sequences obtained by RT-PCR as describedin Example 1 with respect to the HCV Core 201-603-195 hybridoma cellline. Positive PCR products were observed from the heavy chain (H)primer set of C (VH-C) and from the light chain (L) primer sets of B, Cand G (VL-B, VL-C, VL-G). All positive PCR products were gel purified,cloned into the pCR TOPO 2.1 TA vector and transformed into E. coliDH5α. The plasmid DNA was extracted from the E. coli cells and the V_(H)or V_(L) insert confirmed by EcoR I digestion for each PCR product. Thefinal TA clones were selected by sequence alignment as described inExample 1. The sequence results confirmed that VL-G TA clone number 8contained the correct V_(L) sequence (SEQ ID NO:32 encoding apolypeptide as shown in SEQ ID NO:26), while the VH-C TA clone number 13contained the correct V_(H) sequence (SEQ ID NO:31 encoding apolypeptide as shown in SEQ ID NO:25) (see also FIGS. 8A-D).

Cloning the V_(H) and V_(L) Sequences into pBOS Vectors

Using the VL-G TA clone number 8 as the template, a pair of PCR primerswere designed to clone out the mouse V_(L) sequence, namely a HCV NS5Antibody V_(L) 5′-end primer comprising the sequence

5′-GCTCGCGATGCGACATTGTGATGTCACAGT-3′ [SEQ ID NO:27], and a HCV NS5Antibody V_(L) 3′-end primer comprising the sequence

5′-CACCGTACGTTTCAGCTCCAGCTTGGT-3′ [SEQ ID NO:28].

The 5′-end primer contained a partial Kappa signal sequence and a Nru Irestriction site and the 3′-end primer contained a BsiW I restrictionsite. In addition, using the VH-C TA clone number 13 as the template, apair of primers was designed to clone out the mouse V_(H) sequence,namely, a HCV NS5 Antibody V_(H) 5′-end primer comprising the sequence

5′-TACTTCGCGAGAGGTTCAGCTGCAGCAGT-3′ [SEQ ID NO:29], and a HCV NS5Antibody V_(H) 3′-end primer comprising the sequence

5′-TGGTCGACGCTGCAGAGACAGTGACCAG-3′ [SEQ ID NO:30].

For this pair of primers, the 5′-end primer contained a partial heavychain signal sequence and an Nru I restriction site and the 3′-endprimer contained a Sal I restriction site.

The V_(L) and V_(H) sequences were independently amplified by PCR usingthe above primer pairs and the VL-G TA clone number 5 and VH-C TA clonenumber 16, respectively, as the templates. The PCR was executed in 2×Pfxamplification buffer using 15 pmol each of 5′-end and 3′-end primers,1.25 units of Pfx DNA polymerase (Invitrogen) and 100 ng of TA cloneplasmid DNA as template. The PCR comprised 30 cycles of 15 seconds at94° C. followed by 1 minute at 68° C.

The V_(L) and V_(H) PCR products were restriction enzyme trimmed by NruI/BsiW I and Nru I/Sal I digestion, respectively, and then cloned intoeither the pBOS-hck vector (for the V_(L) sequence) or the pBOS-hcgvector (for the V_(H) sequence), as described in Example 1. PlasmidspBOS 48-311-387-L-T4 (containing the V_(L) sequence) and pBOS48-311-387-H-T2 (containing the V_(H) sequence) were selected bysequencing. Once the respective pBOS clones were identified, separate E.coli cell banks containing either the pBOS 48-311-387-L-T4 plasmid orthe pBOS 48-311-387-H-T2 plasmid were made to preserve the pBOS clones.

Chimeric Antibody Functional Testing

pBOS 48-311-387-L-T4 and pBOS 48-311-387-H-T2 plasmid DNA was preparedand transiently transfected into COS 7L cells as described in Example 1.The anti-HCV NS5 chimeric antibody was prepared from the transfected COS7L cells as described in Example 1 and assayed by EIA to confirmreactivity with HCV NS5 antigen (SOD-NS5; Chiron Corporation). The EIAwas conducted as described in Example 1 using the CKS EF HCV NS5 antigenin place of core antigen. The EIA results demonstrated that the chimericantibody from transient expression reactive to the HCV NS5 antigen.

Cloning the V_(H) and V_(L) Sequences into a Stable Expression Vector

The pBOS 48-311-387-L-T4 and pBOS 48-311-387-H-T2 clones were used toconstruct a plasmid clone for stable cell line transfection by firstcloning the sequences into the pBV vector (for the V_(H) sequence) orthe pJV vector (for the V_(L) sequence) and subsequently ligatingtogether the vector fragments obtained from Pac I/Asc I restrictionenzyme digestion as described in Example 1 to provide the pBJ plasmidpBJ HCV NS5 48-311-387 that contains both the V_(H) and the V_(L)sequences. The pBJ clone was screened by Srf I/Not I digestion toconfirm that it contains both sequences. The plasmid map for the finalpBJ clone (pBJ HCV NS5 48-311-387) for the HCV NS5 chimeric antibody isshown in FIG. 7. The V_(H) and V_(L) gene sequences are shown in FIGS.8A-B (SEQ ID NO:31 and 32, respectively). E. coli DH5α cell bankscontaining the pBJ HCV NS5 48-311-387 clone were made.

Establishing a Stable CHO Cell Line and Expression of the HCV ChimericAntibody

The pBJ HCV NS5 48-311-387 plasmid was transfected by calcium phosphatemediated transfection into the DHFR-deficient Chinese Hamster Ovary cellline as described in Example 1. Culture and single cell cloning wasconducted as described in Example 1. When the CHO clones had grown tomore than 50% confluency, the supernatant was tested in an antigenspecific EIA to rank the performance of the CHO clones. The EIA wasconducted as described above. The 6 CHO clones showing the highestsignal were expanded and re-assayed by an antigen specific EIA. 4 CHOclones were selected for single cell cloning using the BD FACSAria flowsorter. When confluent growth was apparent, the cultures were screenedin an EIA that resulted in the selection of 5 clones to be weaned intoserum free medium, including the clone designated CHO 48-311-271. Onefinal subcloning resulted in the selection of HCV NS5 CHO clone48-311-271-455 for cell banking purposes. The cell line was expanded forproduction of chimeric antibodies and for development of a discoverycell bank for storage in liquid nitrogen. The chimeric antibody waspurified from this cell line using standard Protein A purificationprocedures as described on Example 1.

The HCV NS5 CHO 48-311-271-455 cell line was deposited with the AmericanType Culture Collection (ATCC) at 10801 University Boulevard, Manassas,Va. on May 2, 2006 under Patent Deposit Designation PTA-7572.

Example 5 Epitope Mapping of HCV Core Chimeric Monoclonal Antibody

The binding site of the anti-HCV core mouse monoclonal antibodydescribed in Example 1 was mapped to the HCV core protein regioncomprising amino acids 32-36 (GGVYL; SEQ ID NO:33) using syntheticpeptides. Briefly, synthesized short peptides were used to coat thewells of an EIA plate and were blocked with 2% BSA/PBS buffer. Theanti-HCV core mouse monoclonal antibody was added to the EIA plates andincubated for at least 1 hour. The EIA plates were then washed andincubated with goat anti-mouse (“GAM”) HRP labeled antibody for another1 hour. The plates were washed and developed using0-Phenylenediamine-2HCl (OPD), and read at an optical density of 492 nm.

A combination of alanine scanning and yeast display technology weresubsequently used for fine epitope mapping. Briefly, a library ofalanine mutants was constructed using a set of synthetic DNAoligonucleotides which encoded peptides each representing the 27-39region (GGQIVGGVYLLPR; SEQ ID NO:34) of the core antigen and in whicheach individual amino acid in this region was sequentially substitutedby alanine. A wildtype core 27-39 fragment was used as control. The DNAoligonucleotides were cloned into the yeast display vector (pYD41; basedon vector pYD1 available from Invitrogen) at the Nco I and Not Irestriction enzyme sites. Each HCV core alanine mutant was thentransformed into E. coli and plasmid DNA was extracted and sequenced.Final clone selection was based on sequencing. The selected clones weretransformed into EBY100 Saccharomyces cerevesiae cells (Invitrogen).Individual yeast clones were cultured and induced for HCV core peptideexpression. The induced yeast cells were incubated with the HCV corechimeric antibody and a secondary antibody (Alexa Fluor 633 goat antihuman IgG; Invitrogen), and analyzed by Fluorescence Activated CellSorter (FACS Calibur) to determine which alanine mutants had lostantibody binding activity. A loss of antibody binding activity indicatedthat the mutant included an alanine at a position that formed part ofthe epitope for the chimeric antibody. The data showed that HCV coreantigen positions 29 (Gln), 31 (Val), 32 (Gly), 33 (Gly), 36 (Leu) (asshown in FIG. 9A) are binding sites for the HCV core chimeric antibody.

These results confirm that this region set forth in the sequenceQIVGGVYL (SEQ ID NO:100) comprises an epitope for binding to the HCVcore chimeric antibody. The results confirm that for binding to the HCVcore chimeric antibody, these antigenic residues must remain invariantwhile others can be altered, as in the sequence QXVGGXXL, where X is anyamino acid (SEQ ID NO:101), or where X is Ala or Gly (SEQ ID NO:102).

Example 6 Epitope Mapping of HCV NS3 Chimeric Monoclonal Antibody

The HCV NS3 mouse monoclonal antibody produced by hybridoma cell lineanti-HCV NS3 17-903-127 was raised against the region of the HCV NS3antigen spanning amino acids 1192-1457 (265aa). In order to map theepitope that the NS3 chimeric antibody binds within this region, the265aa region was broken up into twelve overlapping fragments byconstructing twelve pairs of DNA oligonucleotides. Specifically, theseoligonucleotides represented the following fragments: 1190-1216;1212-1239; 1235-1257; 1251-1272; 1273-1301; 1297-1323; 1321-1347;1341-1367; 1364-1387; 1388-1414; 1411-1438, and 1434-1459. The pairs ofDNA oligonucleotides were annealed and cloned into yeast display vector(pYD41) at Nco I and Not I restriction enzyme sites. Each of the HCV NS3vectors was transformed into E. coli and plasmid DNA was extracted andsequenced. Final clone selection was based on sequencing. The selectedclones were transformed into EBY100 Saccharomyces cerevesiae cells.Individual yeast clones were cultured and induced for HCV NS3 peptideexpression. The induced yeast cells were incubated with the HCV NS3chimeric antibody and secondary antibody (Alexa Fluor 633 goat antihuman IgG), and analyzed by Fluorescence Activated Cell Sorter (FACSCalibur) to determine which fragment demonstrated positive binding tothe chimeric antibody, thus indicating the epitope location. The datashowed that only HCV NS3 fragment 1190-1216(AKAVDFVPVESLETTMRSPVFTDNSSP; SEQ ID NO:35) demonstrated positivebinding.

Alanine scanning and yeast display technology were then used for fineepitope mapping. A library of alanine mutants representing the 1190-1216region was constructed using a set of synthetic DNA oligonucleotides asgenerally described in Example 5 for the core antigen. Wildtype NS31190-1216 fragment was used as control. Yeast display was carried out asdescribed in Example 5 and the induced yeast cells were incubated withthe HCV NS3 chimeric antibody and a secondary antibody (Alexa Fluor 633goat anti human IgG), and analyzed by Fluorescence Activated Cell Sorter(FACS Calibur) to determine which alanine mutants had lost antibodybinding activity. The data showed that HCV NS3 antigen positions 1194(Asp), 1195 (Phe), 1196 (Val), 1197 (Pro), 1199 (Glu), 1201 (Leu), 1202(Glu), and 1205 (Met) (as shown in FIG. 9B) are binding sites for theHCV NS3 chimeric antibody.

These results confirm that this region set forth in the sequenceDFVPVESLETTM (SEQ ID NO:103) comprises an epitope for binding to the HCVNS3 chimeric antibody. The results confirm that for binding to the HCVNS3 chimeric antibody, these antigenic residues must remain invariantwhile others can be altered, as in the sequence DFVPXEXLEXXM, where X isany amino acid (SEQ ID NO:104), or where X is Ala or Gly (SEQ IDNO:105).

Example 7 Epitope Mapping of HCV NS4 Chimeric Monoclonal Antibody

The HCV NS4 chimeric monoclonal antibody produced by the HCV NS4 CHOE99H6C34sc203 cell line was mapped to the region of the HCV NS4 antigenrepresented by amino acids 1692-1713 (PAIIPDREVLYREFDEMEECSQ; SEQ IDNO:36) using synthetic DNA oligonucleotides and standard yeast displaytechnology, as generally described in Examples 5 and 6. Alanine scanningand yeast display technology were then used for fine epitope mapping. Alibrary of alanine mutants representing the 1692-1713 region wasconstructed using a set of synthetic DNA oligonucleotides as generallydescribed in Example 5 for the core antigen. Wildtype NS4 1692-1713fragment was used as control. Yeast display was carried out as describedin Example 5 and the induced yeast cells were incubated with the HCV NS4chimeric antibody and a secondary antibody (Alexa Fluor 633 goat antihuman IgG), and analyzed by Fluorescence Activated Cell Sorter (FACSCalibur) to determine which alanine mutants had lost antibody bindingactivity. The data showed that HCV NS4 antigen positions 1701 (Leu),1702 (Tyr), 1704 (Gly), 1705 (Phe), 1706 (Asp) (as shown in FIG. 9C) arebinding sites for the HCV NS4 chimeric antibody.

These results confirm that this region set forth in the sequence LYREFD(SEQ ID NO:106) comprises an epitope for binding to the HCV NS4 chimericantibody. The results confirm that for binding to the HCV NS4 chimericantibody, these antigenic residues must remain invariant while otherscan be altered, as in the sequence LYXEFD, where X is any amino acid(SEQ ID NO:107), or where X is Ala or Gly (SEQ ID NO:108).

Example 8 Epitope Mapping of HCV NS5 Chimeric Monoclonal Antibody

The HCV NS5 mouse monoclonal antibody produced by hybridoma cell lineanti-HCV NS5 48-311-387 was raised against the region of the HCV NS5antigen spanning amino acids 2054-2481 (428aa) region. In order to mapthe epitope that the NS5 chimeric antibody binds within this region, the428aa region was broken up into eighteen overlapping fragments byconstructing eighteen pairs of DNA oligonucleotides. Specifically, theseoligonucleotides represented the following fragments: 2048-2076;2075-2101; 2098-2124; 2120-2146; 2144-2177; 2169-2196; 2193-2221;2220-2247; 2245-2272; 2271-2296; 2292-2318; 2313-2339; 2336-2363;2360-2386; 2382-2408; 2408-2436; 2435-2462, and 2457-2486. Epitopemapping utilizing these eighteen pairs of oligonucleotides and the HCVNS5 chimeric antibody was conducted as described in Example 6. The datashowed that only the HCV NS5 fragment 2382-2408(2382-AESYSSMPPLEGEPGDPDLSDGSWSTV-2408; SEQ ID NO:37) demonstratedpositive binding.

Alanine scanning and yeast display technology were then used for fineepitope mapping. A library of alanine mutants representing the 2382-2408region was constructed using a set of synthetic DNA oligonucleotides asgenerally described in Example 5 for the core antigen. Wildtype NS52382-2408 fragment was used as control. Yeast display was carried out asdescribed in Example 5 and the induced yeast cells were incubated withthe HCV NS5 chimeric antibody and a secondary antibody (Alexa Fluor 633goat anti human IgG), and analyzed by Fluorescence Activated Cell Sorter(FACS Calibur) to determine which alanine mutants had lost antibodybinding activity. The data showed that HCV NS5 antigen positions 2390(Pro), 2391 (Leu), 2392 (Glu), 2393 (Gly), 2394 (Glu), 2395 (Pro), 2397(Asp), 2398 (Pro), and 2400 (Leu) (as shown in FIG. 9D) are bindingsites for the HCV NS5 chimeric antibody.

These results confirm that this region set forth in the sequencePLEGEPGDPDL (SEQ ID NO:109) comprises an epitope for binding to the HCVNS5 chimeric antibody. The results confirm that for binding to the HCVNS5 chimeric antibody, these antigenic residues must remain invariantwhile others can be altered, as in the sequence PLEGEPXDPXL, where X isany amino acid (SEQ ID NO:110), or where X is Ala or Gly (SEQ IDNO:111).

Example 9 Determination of the Equilibrium Dissociation Constant (K_(D))for HCV Core Chimeric Monoclonal Antibody

The yeast cells containing wildtype HCV 27-39 core fragment from Example5 were cultured and induced to express the epitope on the cell surface.The induced yeast cells were incubated with varying concentrations ofthe HCV core chimeric antibody (100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM,0.4 nM, 0.14 nM and zero nM). Bound HCV core chimeric antibody was thendetected with goat anti-human IgG conjugated with Alexa 633 fluorophoreusing standard flow cytometeric analysis. The mean florescence intensity(MFI) at each concentration of the HCV core chimeric antibody wasdetermined after excitation of the fluorophore using a flow cytometerequipped with appropriate lasers and detection optics.

The apparent equilibrium dissociation constant (K_(D)) was calculated byplotting MFI vs. [Ag] and determining a best fit curve, wherein thecurve is known to be defined by the formula:Fbkg+Fsat*[Antigen]/(K_(D)+[Antigen]), where Fbkg: background signal andFsat: maxim signal, thereby allowing the determination of K_(D). Theresults indicated that the K_(D) for the HCV core chimeric antibody isabout 0.7 nM.

Example 10 Determination of the Equilibrium Dissociation Constant(K_(D)) for HCV NS3 Chimeric Monoclonal Antibody

The equilibrium dissociation constant (K_(D)) for the HCV NS3 chimericantibody was determined as described in Example 9 using yeast cellscontaining the wildtype HCV 1190-1216 NS3 fragment (from Example 6) andvarying concentrations of the HCV NS3 chimeric antibody (33 nM, 11 nM,3.7 nM, 1.2 nM, 0.4 nM, 0.14 nM, and zero nM). The results indicatedthat the K_(D) for the HCV NS3 chimeric antibody is about 68 nM.

Example 11 Determination of Equilibrium Dissociation Constant (K_(D))for HCV NS4 Chimeric Monoclonal Antibody

The equilibrium dissociation constant (K_(D)) for the HCV NS4 chimericantibody was determined as described in Example 9 using yeast cellscontaining the wildtype HCV 1692-1713 NS4 fragment (from Example 7) andvarying concentrations of the HCV NS4 chimeric antibody (100 nM, 33 nM,11 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0.14 nM, and zero nM). The resultsindicated that the K_(D) for the HCV NS4 chimeric antibody is about 0.5nM.

Example 12 Determination of Equilibrium Dissociation Constant (K_(D))for HCV NS5 Chimeric Monoclonal Antibody

The equilibrium dissociation constant (K_(D)) for the HCV NS5 chimericantibody was determined as described in Example 9 using yeast cellscontaining the wildtype HCV 2382-2408 NS5 fragment (from Example 8) andvarying concentration of the HCV NS5 chimeric antibody (100 nM, 33 nM,11 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0.14 nM, and zero nM). The resultsindicated that the K_(D) for the HCV NS5 chimeric antibody is about 8.8nM.

Example 13 Characterization of HCV Chimeric Antibodies and CHO LinesSecreting Same

The HCV core CHO 201-603-486-333, HCV NS3 CHO 17-903-132sc171, HCV NS4CHO E99H6C34sc203 and HCV NS5 CHO clone 48-311-271-455 cell linesprepared in Examples 1-4 were analyzed for manufacturability, includingcell viability, cell line stability via stable antibody production, andfreedom from adventitious agents via mycoplasma testing. The cell lineswere also tested for monoclonality. The chimeric antibodies werecharacterized by isoelectric focusing (IEF), SDS-PAGE and gel permeationchromatography (GPC).

The results indicated that all the cell lines were >90% clonal and freeof adventitious agents. Analysis of chimeric antibody production overa >3 week time period indicated that all cell lines have a stable orincreasing chimeric antibody production in culture. The average chimericantibody production on a terminal R & D scale (i.e. viable cells werebetween 20-10%) as assessed by HPLC analysis of 3 week old cultures isshown in FIG. 10.

SDS-PAGE gel electrophoresis was performed on all HCV chimericantibodies under reducing condition. About 3 ug of each chimericantibody was mixed with loading buffer with reducing agents, boiled for10 minutes, then loaded onto 12% SDS-PAGE gel and run at 80 Volts for1.5 hours. The heavy chain should migrate between 66.2 kD and 45 kD, andthe light chain should migrate between 31 kD and 21.5 kD. All of the HCVchimeric antibodies showed two distinctive bands by SDS-PAGE thatcorresponded to antibody heavy and light chains.

In order to further characterize the HCV chimeric antibodies,isoelectric focusing (IEF) gel electrophoresis was performed on all fourHCV chimeric antibodies. IEF is a technique that separates proteinsaccording to their net charge. At a given pH, a protein's net chargewill depend its relative number of positive and negative charges. The pHat which the positive charges on a protein equal the negative chargesdefines that protein's isoelectric point (pI). The IEF profiles of thefour HCV chimeric antibodies indicated they have pI values ranging from7.8 to 9.0. Specifically, the pI of the HCV core chimeric antibody wasabout 9.0, the pI of the HCV NS3 antibody was between 8.5 and 9.0, thepI of the HCV NS4 chimeric antibody was between 7.8 and 8.7, and the pIof the HCV NS5 chimeric antibody was between 8.0 and 8.9.

The HCV core chimeric antibodies were also analyzed by gel permeationhigh performance liquid chromatography (GPC-HPLC) on a Waters systemwith a Tosohaas 3000 column. GPC-HPLC is a standard technique used todetermine the purity of a protein or polypeptide as well as itsaggregation state via chromatographic separation based upon molecularsize and shape. Based on three bug injections each of albumin, chimericantibody and gel filtration standard, the final mean purity for eachchimeric antibody lot was calculated. The purity of HCV NS3 CHO17-903-132sc171 lot was 97%. The purity of HCV NS4 CHO E99H6C34sc203 lotwas 58%. The purity of HCV NS5 CHO 48-311-271-455 lot was 90% and thepurity of HCV core CHO 201-603-486-333 lot was 88%.

Example 14 Antigen Reactivity of HCV Chimeric Antibodies in Standard HCVDetection Assays

The four chimeric antibodies (HCV core, NS3, NS4 and NS5) prepared asdescribed in Examples 1-4 were tested using Abbott HCV blood screeningassays on the Prism, AxSYM, ARCHITECT and EIA (Bead) platforms. ThePrism platform assay employs NS3, NS4, NS5 and Core antigens forantibody detection, whereas the AxSYM, ARCHITECT and EIA (Bead) platformassays employ NS3, NS4 and Core antigens for antibody detection. For allplatforms, the antigens are currently qualified using epitope reactiveplasma/sera samples sourced from human donors.

Abbott HCV EIA is an in vitro enzyme immunoassay for qualitativedetection of antibody to Hepatitis C (anti-HCV) in human serum, plasmaor cadaveric serum. The human sample is diluted in a specimen diluentand incubated with a polystyrene bead coated with recombinant HCVantigens. If antibody is present in the sample, immunoglobulins in thepatient sample bind to the antigens coated on the bead. After removingthe unbound materials by washing the bead, human immunoglobulinsremaining bound to the bead are detected by incubating thebead-antigen-antibody complex with a solution containing horseradishperoxidase labeled goat antibodies directed against humanimmunoglobulins using O-phenylenediamine-2HCl (OPD) and reading theintensity of color developed at 492 nm. The final Sample/cutoff (S/Co)are calculated. The cutoff is calculated as follows:

Cutoff value=NCx+(0.25)PCx, where NCx (Negative mean Absorbance)=Totalabsorbance/number of replicates and PCx (Calculation of positivemean)=Total absorbance/number of replicates.

Samples with absorbance values greater than or equal to 0.005 but lessthan the cutoff are considered negative. Samples with absorbance valuesgreater than or equal to the cutoff value are considered initiallyreactive. Following this procedure using the Abbott HCV EIA 2.0 assaykit, the anti-HCV core, NS3 and NS4 chimeric antibodies were allreactive as shown in Table 3. Anti-HCV NS5 chimeric antibody is negativesince there is no HCV NS5 recombinant antigen included in the assay.

The Abbott AXSYM Anti-HCV assay is a microparticle immunoassay (META)for the qualitative detection of anti-HCV IgG to HCV recombinantproteins in human serum or plasma. The AxSYM system used to perform theassays calculates the cutoff rate from the mean rate of two IndexCalibrator replicates and stores the results. The cutoff rate (CO) isdetermined by multiplying the AxSYM anti-HCV Index calibrator mean rateby 0.12.

Samples are considered reactive if the S/CO values are greater than orequal to 1.21. Samples with S/CO values between 0.8 and 1.20 areconsidered to be “greyzone” samples, and samples with S/CO values lessthan 0.79 are considered non-reactive. Following the protocol describedin the Abbott HCV AxSYM Anti-HCV assay package insert, the anti-HCVcore, NS3 and NS4 chimeric antibodies were reactive as shown in Table 3.Again, the anti-HCV NS5 chimeric antibody was negative since there is noHCV NS5 recombinant antigen included in the assay.

The Abbott ARCHITECT Anti-HCV assay is a chemiluminescent microparticleimmunoassay (CMIA) for the qualitative detection of anti-HCV antibodiesin human serum and plasma. The ARCHITECT Anti-HCV assay uses recombinantHCV antigens coated on a microparticle surface to bind antibodies in asample. In the ARCHITECT Anti-HCV final reaction, bound acridinylatedconjugates are used to generate a chemiluminescent signal. The ARCHITECTi System used to perform the assays calculates the cutoff RLU from themean chemiluminescent signal of three Anti-HCV Calibrator 1 replicatesand stores the result. The cutoff RLU is determined by multiplying theAnti-HCV Calibrator 1 mean RLU by 0.074. The ARCHITECT i System thencalculates a result based on the ratio of the sample RLU to the cutoffRLU (S/CO) for each specimen and control.

Samples are considered reactive if the S/CO values are greater than orequal to 1.00. Samples with S/CO values between 0.8 and 0.99 areconsidered to be “greyzone” samples, and samples with S/CO values lessthan 0.79 are considered non-reactive. Following the protocol providedin the Abbott ARCHITECT Anti-HCV assay package insert, the anti-HCVcore, NS3 and NS4 chimeric antibodies are reactive as shown in Table 3.Anti-HCV NS5 chimeric antibody was negative since there is no HCV NS5recombinant antigen included in the assay.

The Abbott Prism HCV assay is a chemiluminescent microparticleimmunoassay for the qualitative detection of anti-HCV antibodies inhuman serum and plasma. The cutoff value is calculated as follows:

Cutoff value=Mean negative calibrator net counts+0.55×Mean Positivecalibrator net counts.

Samples are considered initially reactive if the S/CO values are greaterthan or equal to the cutoff value. Following the protocol provided inthe Abbott Prism HCV assay package insert, the anti-HCV core, NS3, NS4and NS5 chimeric antibodies were all reactive as shown in Table 3.

TABLE 3 Reactivity of Core, NS3, NS4 and NS5 Chimeric Antibody inVarious Abbott HCV Assays^(‡) Con- Sample/Cutoff (S/CO) values Antigencentration EIA ARCHI- Sample Reactivity (mg/mL) (Bead) AxSYM Prism TECTNegative — — 0.2 0.21 0.12 0.05 Control Positive — — 3.21 4.99 1.60 3.63Control^(¶) NS4: 1 NS4 1.41 12.72 31.11 2.58 8.59 NS4: 2 1.90 15.0326.69 2.77 5.88 NS4: 3 2.20 14.30 28.10 2.43 5.41 NS4: 4 1.50 14.0029.68 2.94 8.89 NS4: 5 0.70 13.20 31.35 2.32 7.75 NS4: 6 1.50 13.1533.61 2.67 8.10 NS4: 7 1.19 13.24 33.29 2.66 6.34 Core: 1 Core 1.13 9.0450.89 2.14 11.81 Core: 2 1.00 8.91 47.00 2.14 12.29 Core: 3 0.83 9.0946.37 2.12 14.49 Core: 4 1.80 9.27 46.74 2.08 12.20 Core: 5 2.46 9.2548.67 2.13 12.70 CORE: 6 2.50 9.45 44.70 2.16 12.36 Core: 7 1.90 8.1340.41 1.64 11.17 Core: 8* 0.15 9.04 45.70 1.99 11.31 Core: 9 2.14 8.8544.76 1.92 12.03 Core: 10* 0.01 7.80 4.77 1.24 8.85 NS5: 1 NS5 0.70 0.060.16 2.60 0.05 NS5: 2 1.60 0.03 0.14 2.04 0.06 NS5: 3 1.30 0.04 0.142.38 0.05 NS5: 4 0.87 0.02 0.13 2.59 0.05 NS5: 5 0.58 0.02 0.10 2.550.05 NS3: 1 NS3 3.08 7.44 40.58 3.94 8.75 NS3: 2 2.40 7.58 40.34 4.399.50 NS3: 3 3.70 7.88 38.88 4.41 10.57 NS3: 4 1.90 7.51 37.54 3.89 9.69NS3: 5 2.10 7.71 37.68 4.65 10.38 N53: 6^(#) 1.80 6.77 36.31 4.37 8.70N53: 7^(#) 1.80 7.99 39.64 4.58 9.59 NS3: 8* 0.33 7.30 40.26 4.15 9.52NS3: 9* 0.34 8.00 39.42 4.30 9.61 ^(‡)Chimeric antibodies were tested ata concentration of 500 ng/mL on all Abbott platforms, with the exceptionof the HCV NS5 chimeric antibody on the Abbott Prism, which was testedat a concentration of 10 μg/mL. ^(¶)Positive control consisted of HCVpositive patient plasma samples that was qualified and targeted to aspecific rate or RLU. *Harvest samples, i.e. straight cell culturesupernatant. ^(#)Pre-dialysis samples, i.e. material purified from cellculture supernatant by Protein A purification.

The results show that on the Abbott AxSYM, EIA, Prism and ARCHITECTplatforms the HCV chimeric antibodies demonstrate reactivity to antigensthat is equal to or greater than the human sourced control. In Table 3,the anti-NS5 chimeric antibody does not react on EIA, AxSYM, andARCHITECT assay merely because there is no NS5 antigen coated on thebead/microparticle.

Example 15 Preparation of a Human-Mouse Chimeric Antibody Specific forHepatitis C Virus Core Protein #2

A second anti-core chimeric antibody, HCV Core CHO 14-153-229sc152, wasprepared from the hybridoma 14-153-462. The hybridoma cell line anti-HCVCore 14-153-462 was deposited with the American Type Culture Collection(ATCC) at 10801 University Boulevard, Manassas, Va. on Nov. 21, 2006under Accession No. PTA-8028.

Preparation of a Hybridoma Cell Line

The hybridoma cell line anti-HCV core 14-153-462 was developed asdescribed in Example 1 using the purified HCV recombinant core antigenknown as pL-Core in place of the core CKS-33C-BCD antigen. The pL-CORErecombinant antigen corresponds to amino acids 1-150 of the HCVpolyprotein. The animal immunization regiment utilized one 200 μg boostin the Freunds Adjuvant System, with a pre-fusion boost in saline 2weeks later.

Isolation of mRNA and Identification of Mouse V_(H) and V_(L) Sequences

mRNA was isolated from the hybridoma cell line anti-HCV core 14-153-462and the mouse V_(H) and V_(L) sequences obtained by RT-PCR as describedin Example 1 with respect to the HCV Core 201-603-195 hybridoma cellline. Positive PCR products were observed from the heavy chain (H)primer sets of C (VH-C) and from the light chain (L) primer sets of Band C (VL-B, VL-C). All positive PCR products were gel purified, clonedinto the pCR TOPO 2.1 TA vector and transformed into E. coli. Colony PCRreactions were used to confirm the clone insert. Briefly, the PCR wasconducted using M13 forward and M13 reverse primer (Invitrogen), Taq DNApolymerase and boiled E. coli colony as the template. The PCR compriseddenaturation 1 minute at 94° C.; annealing 1 minute at 50° C. andextension 2 minutes at 72° C., with a final extension 6 minutes at 72°C. for a total of 30 cycles. The clones giving the correct size for thePCR products were grown in LB broth. Each plasmid DNA was purified usingQIAprep spin miniprep Kit (QIAGEN) and sequenced using M13 forward andreverse primers with the Big Dye Terminator v3.1 cycle sequencing kit(Applied Biosystems).

The final TA clone was selected by sequence alignment as described inExample 1. The sequence results confirmed that the VL-B clone containedthe correct V_(L) sequence (SEQ ID NO:41 encoding a polypeptide as shownin SEQ ID NO:39), while the VH-C clones contained the correct V_(H)sequence (SEQ ID NO:40 encoding a polypeptide as shown in SEQ ID NO:38)(see also FIGS. 11A-D).

Cloning the V_(H) and V_(L) Genes into pBOS Vectors

Using the VL-B TA clone number 5 as the template, a pair of PCR primerswere designed to clone out the mouse V_(L) sequence, namely, a HCV coreAntibody V_(L) 5′-end primer

5′-AAATTTTCGCGATGCGACATTGTGCTGACCCAATCTC-3′ [SEQ ID NO:96], and a HCVcore Antibody V_(L)3′-end primer

5′-ACTACTCGTACGTTTGATTTCCAGCTTGGTGCCT-3′ [SEQ ID NO:97].

The 5′-end primer contained a partial Kappa signal sequence and a Nru Irestriction site and the 3′-end primer contained a BsiW I restrictionsite. In addition, using the VH TA clone number 1 as the template, apair of primers was designed to clone out the mouse V_(H) sequence,namely, a HCV core Antibody V_(H) 5′-end primer

5′-AAATTTTCGCGATTTTAAAAGGTGTCCAGTGTCAGATCCA

GTTGGTGCAGTCTGG-3′ [SEQ ID NO:98], and a HCV core Antibody V_(H) 3′-endprimer

5′-TCCTTTGTCGACGCTGAGGAGACGGTGACTGAGGTT-3′ [SEQ ID NO:99].

For this pair of primers, the 5′-end primer contained a partial heavychain signal sequence and an Nru I restriction site and the 3′-endprimer contained a Sal I restriction site.

The V_(L) and V_(H) sequences were independently amplified by PCR usingthe above primer pairs and the VL-B TA clone number 5 and VH-C TA clonenumber 1, respectively, as the templates. The PCR was executed in 1×PCRamplification Buffer using 20 pmol each of the 5′-end and 3′-endprimers, 1 unit of Taq DNA polymerase (Invitrogen), and 2 μl ofbacterial colony as the template. The PCR was performed employing 30cycles of 30 seconds at 94° C. and 30 seconds at 55° C. followed by 1minute at 72° C.

The V_(L) and V_(H) PCR products were restriction enzyme trimmed by NruI/BsiW I and Nri I/Sal I digestion, respectively, and then cloned intoeither the pBOS-hck vector (for the V_(L) sequence) or the pBOS-hcgvector (for the V_(H) sequence), as described in Example 1. PlasmidspBOS 14-153-462-L (containing the V_(L) sequence) and pBOS 14-153-462-H(containing the V_(H) sequence) were selected by sequencing as describedin Example 1. Once the respective pBOS clones were identified, separateE. coli DH5α cell banks containing either the pBOS 14-153-462-L-T0plasmid or the pBOS 14-153-462-H-T0 plasmid were made to preserve thepBOS clones.

Chimeric Antibody Functional Testing

pBOS 14-153-462-L-T0 and pBOS 14-153-462-H-T0 plasmid DNA was preparedand transiently transfected into COS 7L cells as described in Example 1.The anti-HCV core chimeric antibody was prepared from the transfectedCOS 7L cells as described in Example 1 and assayed by EIA to confirmreactivity with HCV core antigen. The EIA was conducted as described inExample 1. The EIA results demonstrated that the chimeric antibody fromtransient expression was reactive to the HCV core antigen.

Cloning the V_(H) and V_(L) Sequences into a Stable Expression Vector

The pBOS 14-153-462-L-T0 and pBOS 14-153-462-H-T0 clones were used toconstruct a plasmid clone for stable cell line transfection by firstcloning the sequences into the pBV vector (for the V_(H) sequence) orthe pJV vector (for the V_(L) sequence) and subsequently ligatingtogether the vector fragments obtained from Pac I/Asc I restrictionenzyme digestion as described in Example 1 to provide the pBJ plasmidpBJ HCV core 14-153-462 that contains both the V_(H) and the V_(L)sequences. The pBJ clone was screened by Srf I/Not I digestion toconfirm that it contains both sequences. The plasmid map for the finalpBJ clone (pBJ HCV core 14-153-462) for the HCV core chimeric antibodyis shown in FIG. 12. The V_(H) and V_(L) gene sequences are shown inFIGS. 11A-B (SEQ ID NO:40 and SEQ ID NO:41, respectively). E. coli DH5αcell banks containing the pBJ 14-153-462-T4 clone were made.

Establishing a Stable CHO Cell Line and Expression of the HCV ChimericAntibody

The pBJ 14-153-462-T4 plasmid was transfected by calcium phosphatemediated transfection into the DHFR-deficient Chinese Hamster Ovary cellline as described in Example 1. Culture and single cell cloning wasconducted as described in Example 1. The 96 well plates were tested byantigen specific EIA and clone number 229 was selected for furtherdevelopment. The EIA was conducted as described above. The HCV core14-153-462 CHO cell clone was cultured and single cell sorted into96-well plates using the BD FACSAria flow cytometer sorter. CHO subclonenumber 152 was selected for weaning into CD-CHO serum free medium(Invitrogen). The final CHO cell line (HCV core 14-153-229sc152) wasexpanded for production of the chimeric antibody and for development ofa discovery cell bank for storage in liquid nitrogen. The chimericantibody was purified from this cell line using standard Protein Apurification procedures as described in Example 1.

The HCV core CHO 14-153-229sc152 cell line was deposited with theAmerican Type Culture Collection (ATCC) at 10801 University Boulevard,Manassas, Va. on May 2, 2006 under Patent Deposit Designation PTA-7571.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification arespecifically incorporated by reference in their entirety to the sameextent as if each such individual patent, publication, and databaseentry were specifically and individually indicated to be incorporated byreference.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention. All such modifications as would be apparent to oneskilled in the art are intended to be included within the scope of thefollowing claims.

1. An immunodiagnostic reagent comprising one or more recombinantantibodies that specifically bind to a diagnostically relevant region ofa hepatitis C virus (HCV) protein, wherein said one or more recombinantantibodies are selected from the group consisting of a chimeric antibodyspecific for HCV core protein, a chimeric antibody specific for HCV E2protein, a chimeric antibody specific for HCV NS3 protein, a chimericantibody specific for HCV NS4 protein, and a chimeric antibody specificfor HCV NS5 protein.
 2. The immunodiagnostic reagent according to claim1, wherein said immunodiagnostic reagent comprises two or more of saidrecombinant antibodies.
 3. The immunodiagnostic reagent according toclaim 1, wherein said immunodiagnostic reagent comprises a chimericantibody selected from the group consisting of: (a) a chimeric antibodyspecific for HCV core protein which specifically binds to an epitopecomprised by the region of the HCV core protein defined by amino acids1-150 of the HCV polyprotein; (b) a chimeric antibody specific for HCVNS3 protein which specifically binds to an epitope comprised by theregion of the HCV NS3 protein defined by amino acids 1192 to 1457 theHCV polyprotein; (c) a chimeric antibody specific for HCV NS4 proteinwhich specifically binds to an epitope comprised by the region of theHCV NS4 protein defined by amino acids 1920 to 1935 or amino acids 1676to 1931 the HCV polyprotein; and (d) a chimeric antibody specific forHCV NS5 protein which specifically binds to an epitope comprised by theregion of the HCV NS5 protein defined by amino acids 2054 to 2995 of theHCV polyprotein.
 4. The immunodiagnostic reagent according to claim 1,wherein said immunodiagnostic reagent comprises a chimeric antibodyselected from the group consisting of: (a) a chimeric antibody specificfor HCV core protein which specifically binds to an epitope comprised bythe amino acid sequence as set forth in SEQ ID NO:34; (b) a chimericantibody specific for HCV NS3 protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:35; (c) a chimeric antibody specific for HCV NS4 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:36; and (d) a chimeric antibody specific for HCVNS5 protein which specifically binds to an epitope comprised by theamino acid sequence as set forth in SEQ ID NO:37.
 5. Theimmunodiagnostic reagent according to claim 1, wherein saidimmunodiagnostic reagent comprises a chimeric antibody selected from thegroup consisting of: (a) a chimeric antibody specific for HCV coreprotein which specifically binds to an epitope comprised by the aminoacid sequence as set forth in SEQ ID NO:100; (b) a chimeric antibodyspecific for HCV core protein which specifically binds to an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:101; and(c) a chimeric antibody specific for HCV core protein which specificallybinds to an epitope comprised by the amino acid sequence as set forth inSEQ ID NO:102.
 6. The immunodiagnostic reagent according to claim 1,wherein said immunodiagnostic reagent comprises a chimeric antibodyselected from the group consisting of: (a) a chimeric antibody specificfor HCV NS3 protein which specifically binds to an epitope comprised bythe amino acid sequence as set forth in SEQ ID NO:103; (b) a chimericantibody specific for HCV NS3 protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:104; and (c) a chimeric antibody specific for HCV NS3 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:105.
 7. The immunodiagnostic reagent according toclaim 1, wherein said immunodiagnostic reagent comprises a chimericantibody selected from the group consisting of: (a) a chimeric antibodyspecific for HCV NS4 protein which specifically binds to an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:106; (b)a chimeric antibody specific for HCV NS4 protein which specificallybinds to an epitope comprised by the amino acid sequence as set forth inSEQ ID NO:107; and (c) a chimeric antibody specific for HCV NS4 proteinwhich specifically binds to an epitope comprised by the amino acidsequence as set forth in SEQ ID NO:108.
 8. The immunodiagnostic reagentaccording to claim 1, wherein said immunodiagnostic reagent comprises achimeric antibody selected from the group consisting of: (a) a chimericantibody specific for HCV NS5 protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:109; (b) a chimeric antibody specific for HCV NS5 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:110; and (c) a chimeric antibody specific for HCVNS5 protein which specifically binds to an epitope comprised by theamino acid sequence as set forth in SEQ ID NO:111.
 9. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(H) region selected from the group consisting of:(a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:1; (b) a V_(H)region comprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:38; (c) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 42, 43 and 44; and(d) a V_(H) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs:48, 49 and
 50. 10. The immunodiagnostic reagentaccording to claim 3, wherein said chimeric antibody comprises a V_(L)region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:2; (b) a V_(L) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:39; (c) a V_(L) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 45, 46 and 47; and (d) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 51, 52 and
 53. 11. The immunodiagnostic reagent according toclaim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:9; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 54, 55 and
 56. 12. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:10; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 57, 58 and
 59. 13. The immunodiagnostic reagent according toclaim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:17; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 60, 43 and
 61. 14. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:18; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 62, 58 and
 63. 15. The immunodiagnostic reagent according toclaim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:25; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 64, 65 and
 66. 16. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:a V_(L) region comprising an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO:26, and a V_(L) regioncomprising complementarity determining region sequences substantiallyidentical to one or more of the sequences set forth in SEQ ID NOs: 67,46 and
 68. 17. The immunodiagnostic reagent according to claim 1,wherein said immunodiagnostic reagent is a reagent selected from thegroup consisting of a detection reagent, a standardization reagent, anda positive control reagent.
 18. A chimeric antibody which specificallybinds to a diagnostically relevant region of a HCV protein selected fromthe group consisting of HCV core protein, HCV NS3 protein, HCV NS4protein, and HCV NS5 protein.
 19. The chimeric antibody according toclaim 18, wherein said chimeric antibody is selected from the groupconsisting of: (a) a chimeric antibody specific for HCV core proteinwhich specifically binds to an epitope comprised by the region of theHCV core protein defined by amino acids 1-150 of the HCV polyprotein;(b) a chimeric antibody specific for HCV NS3 protein which specificallybinds to an epitope comprised by the region of the HCV NS3 proteindefined by amino acids 1192 to 1457 the HCV polyprotein; (c) a chimericantibody specific for HCV NS4 protein which specifically binds to anepitope comprised by the region of the HCV NS4 protein defined by aminoacids 1920 to 1935 or amino acids 1676 to 1931 the HCV polyprotein; and(d) a chimeric antibody specific for HCV NS5 protein which specificallybinds to an epitope comprised by the region of the HCV NS5 proteindefined by amino acids 2054 to 2995 of the HCV polyprotein.
 20. Thechimeric antibody according to claim 18, wherein said chimeric antibodyis selected from the group consisting of: (a) a chimeric antibodyspecific for HCV core protein which specifically binds to an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:34; (b) achimeric antibody specific for HCV NS3 protein which specifically bindsto an epitope comprised by the amino acid sequence as set forth in SEQID NO:35; (c) a chimeric antibody specific for HCV NS4 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:36; and (d) a chimeric antibody specific for HCVNS5 protein which specifically binds to an epitope comprised by theamino acid sequence as set forth in SEQ ID NO:37.
 21. The chimericantibody according to claim 18, wherein said chimeric antibody isselected from the group consisting of: (a) a chimeric antibody specificfor HCV core protein which specifically binds to an epitope comprised bythe amino acid sequence as set forth in SEQ ID NO:100; (b) a chimericantibody specific for HCV core protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:101; and (c) a chimeric antibody specific for HCV core protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:102.
 22. The chimeric antibody according to claim18, wherein said chimeric antibody is selected from the group consistingof: (a) a chimeric antibody specific for HCV NS3 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:103; (b) a chimeric antibody specific for HCV NS3protein which specifically binds to an epitope comprised by the aminoacid sequence as set forth in SEQ ID NO:104; and (c) a chimeric antibodyspecific for HCV NS3 protein which specifically binds to an epitopecomprised by the amino acid sequence as set forth in SEQ ID NO:105. 23.The chimeric antibody according to claim 18, wherein said chimericantibody is selected from the group consisting of: (a) a chimericantibody specific for HCV NS4 protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:106; (b) a chimeric antibody specific for HCV NS4 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:107; and (c) a chimeric antibody specific for HCVNS4 protein which specifically binds to an epitope comprised by theamino acid sequence as set forth in SEQ ID NO:108.
 24. The chimericantibody according to claim 18, wherein said chimeric antibody isselected from the group consisting of: (a) a chimeric antibody specificfor HCV NS5 protein which specifically binds to an epitope comprised bythe amino acid sequence as set forth in SEQ ID NO:109; (b) a chimericantibody specific for HCV NS5 protein which specifically binds to anepitope comprised by the amino acid sequence as set forth in SEQ IDNO:110; and (c) a chimeric antibody specific for HCV NS5 protein whichspecifically binds to an epitope comprised by the amino acid sequence asset forth in SEQ ID NO:111.
 25. The chimeric antibody according to claim19, wherein said chimeric antibody comprises a V_(H) region selectedfrom the group consisting of: (a) a V_(H) region comprising an aminoacid sequence substantially identical to the sequence as set forth inSEQ ID NO:1; (b) a V_(H) region comprising an amino acid sequencesubstantially identical to the sequence as set forth in SEQ ID NO:38;(c) a V_(H) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 42, 43 and 44; and (d) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs:48, 49 and
 50. 26.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:2; (b) a V_(L)region comprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:39; (c) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 45, 46 and 47; and(d) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 51, 52 and
 53. 27. The chimeric antibody accordingto claim 19, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:9; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 54, 55 and
 56. 28. The chimericantibody according to claim 19, wherein said chimeric antibody comprisesa V_(L) region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:10; and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 57, 58 and
 59. 29.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(H) region selected from the group consisting of:(a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:17; and (b) a V_(H)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 60, 43 and
 61. 30. The chimeric antibody according to claim 19,wherein said chimeric antibody comprises a V_(L) region selected fromthe group consisting of: (a) a V_(L) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO:18; and (b) a V_(L) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesset forth in SEQ ID NOs: 62, 58 and
 63. 31. The chimeric antibodyaccording to claim 19, wherein said chimeric antibody comprises a V_(H)region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:25; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs:64, 65 and
 66. 32.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:26; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 67, 46 and
 68. 33. The chimeric antibody according to claim 19,wherein said chimeric antibody is substantially identical to thechimeric antibody expressed by a cell line selected from the groupconsisting of HCV core CHO 201-603-486-333, HCV core CHO14-153-229sc152, HCV NS3 CHO 17-903-132sc171, HCV NS4 CHO E99H6C34sc203,and HCV NS5 CHO 48-311-271-455.
 34. A mouse monoclonal antibodyexpressed by a cell line selected from the group consisting of anti-HCVCore 201-603-195, anti-HCV Core 14-153-462, anti-HCV NS3 17-903-127,anti-HCV NS4 E99H6C34, and anti-HCV NS5 48-311-387.
 35. A cell linewhich expresses a chimeric antibody that specifically binds to adiagnostically relevant region of a hepatitis C virus protein, whereinsaid cell line is selected from the group consisting of HCV core CHO201-603-486-333, HCV core CHO 14-153-229sc152, HCV NS3 CHO17-903-132sc171, HCV NS4 CHO E99H6C34sc203, and HCV NS5 CHO48-311-271-455.
 36. A cell line which expresses a mouse monoclonalantibody that specifically binds to a diagnostically relevant region ofa hepatitis C virus protein, wherein said cell line is selected from thegroup consisting of anti-HCV Core 201-603-195, anti-HCV Core 14-153-462,anti-HCV NS3 17-903-127, anti-HCV NS4 E99H6C34, and anti-HCV NS548-311-387.
 37. A method of standardizing a HCV antibody detection assaycomprising employing as a sensitivity panel the immunodiagnostic reagentaccording to claim
 1. 38. A method for detecting the presence of HCVantigens comprising the steps of: (a) obtaining a test sample; (b)contacting said test sample with the immunodiagnostic reagent accordingto claim 1 under conditions that allow formation of a chimericantibody:antigen complex; and (b) detecting any chimericantibody:antigen complex formed as indicating the presence of said HCVantigens.
 39. A method for detecting the presence of HCV antibodiescomprising the steps of: (a) obtaining a test sample; (b) contactingsaid test sample with one or more HCV antigens specific for the HCVantibodies under conditions that allow formation of an antigen:antibodycomplex; and (c) detecting the any antigen:antibody complex formed asindicating the presence of said HCV antigens, wherein the improvementcomprises wherein the immunodiagnostic reagent according to claim 1 isemployed in said method as a positive control or standardizationreagent.
 40. A diagnostic kit for the detection of hepatitis C viruscomprising the immunodiagnostic reagent of claim
 1. 41. A method ofidentifying amino acid residues within an HCV protein epitope that arebound by an antibody that specifically binds to said HCV proteinepitope, said method comprising the steps of: (a) obtaining a yeastdisplay library comprising a series of peptides displayed on the surfaceof host cells (i.e., yeast cells), wherein said series of yeastdisplayed peptides comprise amino acid sequences corresponding to theamino acid sequence of said epitope in which each individual amino acidof said epitope has been sequentially substituted by alanine; (b)contacting said yeast display library with an antibody that specificallybinds to said epitope under conditions that permit binding of saidantibody to said epitope; and (c) identifying those peptides displayedon yeast cells that are not bound by said antibody in step (b), whereinan absence of antibody binding indicates that the yeast displayedpeptide contains an alanine residue at an amino acid position bound bysaid antibody in the epitope.
 42. The immunodiagnostic reagent accordingto claim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:7; (b) a V_(H) region comprising an amino acid sequencesubstantially identical to the sequence encoded by SEQ ID NO:40; (c) aV_(H) region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 69, 70 and 71; and (d) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences encoded by SEQ ID NOs:75, 76 and
 77. 43. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:8; (b) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:41; (c) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 72, 73 and 74; and(d) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 78, 79 and
 80. 44. The immunodiagnostic reagentaccording to claim 3, wherein said chimeric antibody comprises a V_(H)region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:15; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 81, 82 and
 83. 45.The immunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:16; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 84, 85 and
 86. 46. The immunodiagnostic reagent according toclaim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:23; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences encoded by SEQ ID NOs: 87, 70 and
 88. 47. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:24; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 89, 85 and
 90. 48. The immunodiagnostic reagent according toclaim 3, wherein said chimeric antibody comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:31; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences encoded by SEQ ID NOs: 91, 92 and
 93. 49. Theimmunodiagnostic reagent according to claim 3, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:a V_(L) region comprising an amino acid sequence substantially identicalto the sequence encoded by SEQ ID NO:32, and a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 94, 73 and
 95. 50.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(H) region selected from the group consisting of:(a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:7; (b) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:40; (c) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 69, 70 and 71; and(d) a V_(H) region comprising complementarity determining regionsequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs:75, 76 and
 77. 51. The chimeric antibody accordingto claim 19, wherein said chimeric antibody comprises a V_(L) regionselected from the group consisting of: (a) a V_(L) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:8; (b) a V_(L) region comprising an amino acid sequencesubstantially identical to the sequence encoded by SEQ ID NO:41; (c) aV_(L) region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 72, 73 and 74; and (d) a V_(L) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences encoded by SEQ ID NOs: 78, 79 and
 80. 52. The chimericantibody according to claim 19, wherein said chimeric antibody comprisesa V_(H) region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:15; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 81, 82 and
 83. 53.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(L) region selected from the group consisting of:(a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:16; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 84, 85 and
 86. 54. The chimeric antibody according to claim 19,wherein said chimeric antibody comprises a V_(H) region selected fromthe group consisting of: (a) a V_(H) region comprising an amino acidsequence substantially identical to the sequence encoded by SEQ IDNO:23; and (b) a V_(H) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 87, 70 and
 88. 55. The chimeric antibodyaccording to claim 19, wherein said chimeric antibody comprises a V_(L)region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:24; and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 89, 85 and
 90. 56.The chimeric antibody according to claim 19, wherein said chimericantibody comprises a V_(H) region selected from the group consisting of:(a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:31; and (b) a V_(H)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 91, 92 and
 93. 57. The chimeric antibody according to claim 19,wherein said chimeric antibody comprises a V_(L) region selected fromthe group consisting of: (a) a V_(L) region comprising an amino acidsequence substantially identical to the sequence encoded by SEQ IDNO:32, and (b) a V_(L) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 94, 73 and
 95. 58. An isolated polypeptidecomprising a portion of a chimeric antibody that specifically binds to adiagnostically relevant region of a HCV protein selected from the groupconsisting of HCV core protein, HCV NS3 protein, HCV NS4 protein, andHCV NS5 protein.
 59. The isolated polypeptide according to claim 58,wherein said chimeric antibody is selected from the group consisting of:(a) a chimeric antibody specific for HCV core protein which specificallybinds to an epitope comprised by the region of the HCV core proteindefined by amino acids 1-150 of the HCV polyprotein; (b) a chimericantibody specific for HCV NS3 protein which specifically binds to anepitope comprised by the region of the HCV NS3 protein defined by aminoacids 1192 to 1457 the HCV polyprotein; (c) a chimeric antibody specificfor HCV NS4 protein which specifically binds to an epitope comprised bythe region of the HCV NS4 protein defined by amino acids 1920 to 1935 oramino acids 1676 to 1931 the HCV polyprotein; and (d) a chimericantibody specific for HCV NS5 protein which specifically binds to anepitope comprised by the region of the HCV NS5 protein defined by aminoacids 2054 to 2995 of the HCV polyprotein.
 60. The isolated polypeptideaccording to claim 59, wherein said polypeptide comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:1; (b) a V_(H) region comprising an amino acid sequencesubstantially identical to the sequence as set forth in SEQ ID NO:38;(c) a V_(H) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 42, 43 and 44; and (d) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs:48, 49 and
 50. 61.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(L) region selected from the group consisting of: (a) aV_(L) region comprising an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO:2; (b) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:39; (c) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 45, 46 and 47; and(d) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 51, 52 and
 53. 62. The isolated polypeptideaccording to claim 59, wherein said polypeptide comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:9; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 54, 55 and
 56. 63. The isolatedpolypeptide according to claim 59, wherein said polypeptide comprises aV_(L) region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:10; and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 57, 58 and
 59. 64.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(H) region selected from the group consisting of: (a) aV_(H) region comprising an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO:17; and (b) a V_(H) regioncomprising complementarity determining region sequences substantiallyidentical to one or more of the sequences set forth in SEQ ID NOs: 60,43 and
 61. 65. The isolated polypeptide according to claim 59, whereinsaid polypeptide comprises a V_(L) region selected from the groupconsisting of: (a) a V_(L) region comprising an amino acid sequencesubstantially identical to the sequence as set forth in SEQ ID NO:18;and (b) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 62, 58 and
 63. 66. The isolated polypeptideaccording to claim 59, wherein said polypeptide comprises a V_(H) regionselected from the group consisting of: (a) a V_(H) region comprising anamino acid sequence substantially identical to the sequence as set forthin SEQ ID NO:25; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs:64, 65 and
 66. 67. The isolatedpolypeptide according to claim 59, wherein said polypeptide comprises aV_(L) region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:26; and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 67, 46 and
 68. 68.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(H) region selected from the group consisting of: (a) aV_(H) region comprising an amino acid sequence substantially identicalto the sequence encoded by SEQ ID NO:7; (b) a V_(H) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:40; (c) a V_(H) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 69, 70 and 71; and (d) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs:75, 76 and
 77. 69.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(L) region selected from the group consisting of: (a) aV_(L) region comprising an amino acid sequence substantially identicalto the sequence encoded by SEQ ID NO:8; (b) a V_(L) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:41; (c) a V_(L) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 72, 73 and 74; and (d) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 78, 79 and
 80. 70.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(H) region selected from the group consisting of: (a) aV_(H) region comprising an amino acid sequence substantially identicalto the sequence encoded by SEQ ID NO:15; and (b) a V_(H) regioncomprising complementarity determining region sequences substantiallyidentical to one or more of the sequences encoded by SEQ ID NOs: 81, 82and
 83. 71. The isolated polypeptide according to claim 59, wherein saidpolypeptide comprises a V_(L) region selected from the group consistingof: (a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:16; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 84, 85 and
 86. 72. The isolated polypeptide according to claim59, wherein said polypeptide comprises a V_(H) region selected from thegroup consisting of: (a) a V_(H) region comprising an amino acidsequence substantially identical to the sequence encoded by SEQ IDNO:23; and (b) a V_(H) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 87, 70 and
 88. 73. The isolated polypeptideaccording to claim 59, wherein said polypeptide comprises a V_(L) regionselected from the group consisting of: (a) a V_(L) region comprising anamino acid sequence substantially identical to the sequence encoded bySEQ ID NO:24; and (b) a V_(L) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences encoded by SEQ ID NOs: 89, 85 and
 90. 74. The isolatedpolypeptide according to claim 59, wherein said polypeptide comprises aV_(H) region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:31; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 91, 92 and
 93. 75.The isolated polypeptide according to claim 59, wherein said polypeptidecomprises a V_(L) region selected from the group consisting of: (a) aV_(L) region comprising an amino acid sequence substantially identicalto the sequence encoded by SEQ ID NO:32, and (b) a V_(L) regioncomprising complementarity determining region sequences substantiallyidentical to one or more of the sequences encoded by SEQ ID NOs: 94, 73and
 95. 76. An isolated polynucleotide encoding a portion of a chimericantibody that specifically binds to a diagnostically relevant region ofa HCV protein selected from the group consisting of HCV core protein,HCV NS3 protein, HCV NS4 protein, and HCV NS5 protein.
 77. The isolatedpolynucleotide according to claim 76, wherein said chimeric antibody isselected from the group consisting of: (a) a chimeric antibody specificfor HCV core protein which specifically binds to an epitope comprised bythe region of the HCV core protein defined by amino acids 1-150 of theHCV polyprotein; (b) a chimeric antibody specific for HCV NS3 proteinwhich specifically binds to an epitope comprised by the region of theHCV NS3 protein defined by amino acids 1192 to 1457 the HCV polyprotein;(c) a chimeric antibody specific for HCV NS4 protein which specificallybinds to an epitope comprised by the region of the HCV NS4 proteindefined by amino acids 1920 to 1935 or amino acids 1676 to 1931 the HCVpolyprotein; and (d) a chimeric antibody specific for HCV NS5 proteinwhich specifically binds to an epitope comprised by the region of theHCV NS5 protein defined by amino acids 2054 to 2995 of the HCVpolyprotein.
 78. The isolated polynucleotide according to claim 77,wherein said polynucleotide encodes a V_(H) region selected from thegroup consisting of: (a) a V_(H) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO:1; (b) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:38; (c) a V_(H)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 42, 43 and 44; and (d) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs:48, 49 and
 50. 79. The isolatedpolynucleotide according to claim 77, wherein said polynucleotideencodes a V_(L) region selected from the group consisting of: (a) aV_(L) region comprising an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO:2; (b) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:39; (c) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 45, 46 and 47; and(d) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequences setforth in SEQ ID NOs: 51, 52 and
 53. 80. The isolated polynucleotideaccording to claim 77, wherein said polynucleotide encodes a V_(H)region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:9; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 54, 55 and
 56. 81.The isolated polynucleotide according to claim 77, wherein saidpolynucleotide encodes a V_(L) region selected from the group consistingof: (a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in SEQ ID NO:10; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences set forth in SEQID NOs: 57, 58 and
 59. 82. The isolated polynucleotide according toclaim 77, wherein said polynucleotide encodes a V_(H) region selectedfrom the group consisting of: (a) a V_(H) region comprising an aminoacid sequence substantially identical to the sequence as set forth inSEQ ID NO:17; and (b) a V_(H) region comprising complementaritydetermining region sequences substantially identical to one or more ofthe sequences set forth in SEQ ID NOs: 60, 43 and
 61. 83. The isolatedpolynucleotide according to claim 77, wherein said polynucleotideencodes a V_(L) region selected from the group consisting of: (a) aV_(L) region comprising an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO:18; and (b) a V_(L) regioncomprising complementarity determining region sequences substantiallyidentical to one or more of the sequences set forth in SEQ ID NOs: 62,58 and
 63. 84. The isolated polynucleotide according to claim 77,wherein said polynucleotide encodes a V_(H) region selected from thegroup consisting of: (a) a V_(H) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO:25; and (b) a V_(H) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesset forth in SEQ ID NOs:64, 65 and
 66. 85. The isolated polynucleotideaccording to claim 77, wherein said polynucleotide encodes a V_(L)region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence as set forth in SEQ ID NO:26; and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences set forth in SEQ ID NOs: 67, 46 and
 68. 86.The isolated polynucleotide according to claim 77, wherein saidpolynucleotide encodes a V_(H) region selected from the group consistingof: (a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:7; (b) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:40; (c) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 69, 70 and 71; and(d) a V_(H) region comprising complementarity determining regionsequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs:75, 76 and
 77. 87. The isolated polynucleotideaccording to claim 77, wherein said polynucleotide encodes a V_(L)region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:8; (b) a V_(L) region comprising an aminoacid sequence substantially identical to the sequence encoded by SEQ IDNO:41; (c) a V_(L) region comprising complementarity determining regionsequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 72, 73 and 74; and (d) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 78, 79 and
 80. 88.The isolated polynucleotide according to claim 77, wherein saidpolynucleotide encodes a V_(H) region selected from the group consistingof: (a) a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:15; and (b) a V_(H)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 81, 82 and
 83. 89. The isolated polynucleotide according toclaim 77, wherein said polynucleotide encodes a V_(L) region selectedfrom the group consisting of: (a) a V_(L) region comprising an aminoacid sequence substantially identical to the sequence encoded by SEQ IDNO:16; and (b) a V_(L) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 84, 85 and
 86. 90. The isolated polynucleotideaccording to claim 77, wherein said polynucleotide encodes a V_(H)region selected from the group consisting of: (a) a V_(H) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:23; and (b) a V_(H) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 87, 70 and
 88. 91.The isolated polynucleotide according to claim 77, wherein saidpolynucleotide encodes a V_(L) region selected from the group consistingof: (a) a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by SEQ ID NO:24; and (b) a V_(L)region comprising complementarity determining region sequencessubstantially identical to one or more of the sequences encoded by SEQID NOs: 89, 85 and
 90. 92. The isolated polynucleotide according toclaim 77, wherein said polynucleotide encodes a V_(H) region selectedfrom the group consisting of: (a) a V_(H) region comprising an aminoacid sequence substantially identical to the sequence encoded by SEQ IDNO:31; and (b) a V_(H) region comprising complementarity determiningregion sequences substantially identical to one or more of the sequencesencoded by SEQ ID NOs: 91, 92 and
 93. 93. The isolated polynucleotideaccording to claim 77, wherein said polynucleotide encodes a V_(L)region selected from the group consisting of: (a) a V_(L) regioncomprising an amino acid sequence substantially identical to thesequence encoded by SEQ ID NO:32, and (b) a V_(L) region comprisingcomplementarity determining region sequences substantially identical toone or more of the sequences encoded by SEQ ID NOs: 94, 73 and 95.